Systems and methods for analyzing bodily fluids

ABSTRACT

This invention is in the field of medical devices. Specifically, the present invention provides portable medical devices that allow real-time detection of analytes from a biological fluid. The methods and devices are particularly useful for providing point-of-care testing for a variety of medical applications.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 12/576,197, filed on Oct. 8, 2009, which is a continuation of U.S.application Ser. No. 11/389,409, filed on Mar. 24, 2006, now U.S. Pat.No. 7,635,594, which claims the benefit of U.S. Provisional ApplicationNo. 60/678,801, filed May 9, 2005 and U.S. Provisional Application No.60/705,489, filed Aug. 5, 2005 and U.S. Provisional Application No.60/717,192, filed Sep. 16, 2005, and U.S. Provisional Application No.60/721,097, filed Sep. 28, 2005, all of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

This invention is in the field of medical devices. Specifically, thepresent invention provides portable medical devices that allow real-timedetection of analytes from a biological fluid. The methods and devicesare particularly useful for providing point-of-care testing for avariety of medical applications.

BACKGROUND OF THE INVENTION

The discovery of a vast number of disease biomarkers and theestablishment of miniaturized microfluidic systems have opened up newavenues to devise methods and systems for the prediction, diagnosis andtreatment of diseases in a point-of-care setting. Point-of-care testingis particularly desirable because it rapidly delivers results to medicalpractitioners and enables faster consultation. Early diagnosis allows apractitioner to begin treatment sooner and thus avoiding unattendeddeterioration of a patient's condition. Examples of point-of-careanalyses include tests for glucose, drugs of abuse, serum cholesterol,pregnancy, and ovulation. However, these and other currently availablepoint-of-care methods and systems do not provide an integrated solutionfor sample acquisition, testing, analysis and communication of resultsto medical practitioners or health providers when needed. Thus, thereremains a considerable need for a portable, multi-parameter measurementinstrument that provides convenient and rapid data collection,transmission, analysis, as well as on-line medical consultation ordecision making.

New and improved point-of-care testing is also needed for research anddevelopment of therapeutic agents as well as for monitoring possibleadverse drug reactions (ADRs), after a drug is brought to the marketplace.

The safety and efficacy of a drug is determined by the pharmacokinetic(what the body does to the drug) and pharmacodynamic parameters (whatthe drug does to the body) of the drug. Currently, the pharmacokinetic(PK) and pharmacodymanic (PD) parameters of a drug are generallydetermined by first drawing blood samples from a patient followed bylaboratory analyses. Such approach has numerous shortcomings. First, thepatient is generally required to visit a clinic to provide clinicalsamples such as blood or urine samples at multiple time points. Second,most of the analytical techniques for determining target analyte andbiomarker concentrations that reflect either the pharmacokinetic (PK)and pharmacodymanic (PD) parameters require that the blood samples bepre-processed before the parameters can be determined. This results indelay of data response, variability in physiological drug distributionand metabolism (warranting poor dosing), sparse sampling, and the lackof dosing history. Notably, numerous clinical trials often suffer frominsufficient numbers of blood tests because of poor patient compliance;the patients often fail to return to a phlebotomist to provide the bloodsamples required by the trial.

Similarly, the current techniques and systems for monitoring ADRs arealso inadequate. ADRs are one of the leading causes of morbidity andmortality in health care. The Institute of Medicine reported in January2000 that 44,000 to 98,000 deaths occurred due to medical errors, ofwhich 7,000 deaths were due to ADRs. Other studies conducted onhospitalized patient populations have indicated an ever higher overallincidence of several ADRs. Several reasons contribute to the prevalenceof ADRs. First, there are more combination therapies available topatients. Second, there is an increasing trend towards chronic use ofdrugs (statins such as Lipitor and Cox-2 inhibitors such as Vioxx).Chronic use of drugs also increases the chance that changes in thepatient's lifestyle, health status and use of other medications willoccur. In women, the chronic use of drugs can result in unanticipatedconsequences if the woman becomes pregnant. Such risks are of particularconcern to the fetus, which is especially susceptible to ADRs includingteratogenicity.

A further important factor in managing the risks and benefits of drugtherapy is patient compliance. Patients often fail to take scheduleddose of drug, take more than the prescribed dose, or fail to complete acourse of drug therapy (especially common in treatment for infectiousdisease). These behaviors (deliberate or inadvertent) result in improperlevels of drugs in the body which can cause serious adverse effects. Thepatient is typically oblivious to such consequences and the prescribingphysician is also unlikely to realize the problem before severalconsequences occur.

Thus, there remains a pressing need for methods and apparatus that allowreal-time data transmission between patient and medical practitioners toenable efficient communication and high throughput point-of-care testingin an ambulatory context. A beneficial system will detect ADRs, andefficacy and/or toxicity of a therapeutic agent in real-time in anambulatory setting. It may also facilitate medical practitionersassessing patients' physiological conditions in response to therapeuticagents during the course of clinical trials or follow-on treatments. Thepresent invention satisfies these needs and provides related advantagesas well.

SUMMARY OF THE INVENTION

One aspect of the present invention is the design of a system capable ofproviding real-time data transmission between a patient and medicalpractitioners to facilitate high throughput point-of-care testing in anambulatory setting. The systems and methods provided herein simplify thelaborious and expensive procedures of processing and analyzing thesamples collected from a subject (e.g., a patient) without the use oflaboratory equipment or facility. The systems and methods areparticularly useful for detection of an analyte from a small sample ofbodily fluid to effect diagnosis, prognosis, treatment, and developmentof therapeutics.

Accordingly, in one embodiment, the present invention provides a systemfor detecting an analyte in a bodily fluid from a subject. The systemcomprises a) a fluidic device, said fluidic device comprising a samplecollection unit and an assay assembly, wherein said sample collectionunit allows a sample of bodily fluid of less than 500 ul to react withreactants contained within said assay assembly to yield a detectablesignal indicative of the presence of said analyte collected in saidsample of bodily fluid; b) a reader assembly comprising a detectionassembly for detecting said detectable signal; and c) a communicationassembly for transmitting said detected signal to an external device.

In another embodiment, the present invention provides a systemcomprising a fluidic device. The fluidic device comprises the followingelements: a) a sample collection unit and an assay assembly, whereinsaid sample collection unit allows a sample of bodily fluid to reactwith reactants contained within said assay assembly based on a protocoltransmitted from an external device to yield a detectable signalindicative of the presence of said analyte; b) a reader assemblycomprising a detection assembly for detecting said detectable signal;and c) a communication assembly for transmitting said detected signal toan external device.

In one aspect, the system employs a protocol transmitted from anexternal device, preferably through a wirelessly device such as a cellphone. In another aspect, the fluidic device further comprises anidentifier to provide the identity of said fluidic device that isadapted to trigger the transmission of the protocol. Where desired, theprotocol may vary depending on the identify of said fluidic device thatis recognizable by an identifier detector.

The present invention also provides a method of using the systems andother devices provided herein. In one embodiment, the present inventionprovides a method for detecting an analyte in a bodily fluid of asubject. The method involves the steps of a) providing the subjectsystem, b) allowing a sample of bodily fluid to react with the reactantscontained within said assay assembly to yield a detectable signalindicative of the presence of said analyte; and c) detecting saiddetectable signal. Where desired, the method may further comprise thestep of quantifying the amount of said analyte present in said bodilyfluid. The method may also comprise the step of comparing the amount ofsaid analyte present in said biologic fluid to a predetermined amount ofsaid analyte. Also optionally included in the method is taking a medicalaction when the amount of said analyte present in said bodily fluid isstatistically different than said predetermined amount. The medicalaction may involve notifying a pharmacy that a prescription for suchsubject needs to be altered.

The present invention further provides a system for monitoring more thanone pharmacological parameter useful for assessing efficacy and/ortoxicity of a therapeutic agent. The system typically comprises a) afluidic device comprising a cartridge, said cartridge comprising atleast one sample collection unit and an assembly; wherein said samplecollection unit allows a sample of bodily fluid comprising a pluralityof analytes indicative or said more than one pharmacological parameterto react with reactants contained within said assay assembly, saidreaction yields detectable signals indicative of the values of the morethan one pharmacological parameter from said sample of bodily fluid; b)a reader assembly comprising a detection assembly for detecting saiddetectable signals; and c) a communication assembly for transmittingsaid detected signals to an external device.

The present invention also provides a method of using such system. Ingeneral, the method involves the steps of a) subjecting a sample ofbodily fluid from a subject administered with the pharmaceutical agentto a fluidic device for profiling said more than one pharmacologicalparameter, said fluidic medical device comprising a cartridge, saidcartridge comprising at least one sample collection unit, and an assayassembly comprising reaction reagents; b) actuating said fluidic deviceand directing said immunoassay reagents within said fluidic device; c)allowing said sample of bodily fluid to react with immunoassay reagentsto yield detectable signals indicative of the values of the more thanone pharmacological parameter from said sample; and d) detecting saiddetectable signal generated from said sample of bodily fluid.

Further provided in the present invention is a method of automaticallymonitoring patient compliance with a medical treatment involving atherapeutic agent. The method involves a) providing a sample of bodilyfluid from said patient; b) allowing the sample of bodily fluid to reactwith assay reagents in a fluidic device to detect an analyte indicativeof compliance or non-compliance of the medical treatment; c) detect thepresence or absence of the analyte; and d) notifying said patient or amedical practitioner of said compliance or noncompliance

Also included is a business method of assisting a clinician in providingan individualized medical treatment. The method involves the steps of a)collecting at least one pharmacological parameter from an individualreceiving a medication, said collecting step is effected by subjecting asample of bodily fluid to reactants contained in a fluidic device, whichis provided to said individual to yield a detectable signal indicativeof said at least one pharmacological parameter; b) cross referencingwith the aid of a computer medical records of said individual with theat least one pharmacological parameter of said individual, therebyassisting said clinician in providing individualized medical treatment.

The present invention provides a business method of monitoring aclinical trial of a pharmaceutical agent. The method typically comprisesthe steps of a) collecting at least one pharmacological parameter from asubject in said clinical trial at a plurality of time intervals, saidcollecting step is effected at each time interval by subjecting a sampleof bodily fluid from said subject to reactants contained in a fluidicdevice, wherein said fluidic device is provided to said subject to yielddetectable signals indicative of the values of said at least onepharmacological parameter at a plurality of time intervals; b) comparingthe detected values to a threshold value predetermined for saidpharmacological parameter; c) notifying a clinician and/or a sponsorinvolved in said clinical trial when a statistically significantdiscrepancy exists between the detected values and the threshold value.

In a separate embodiment, the present invention further provides amethod of obtaining pharmacological data useful for assessing efficacyand/or toxicity of a therapeutical agent from a test animal. The methodtypically involves the steps of a) providing a fluidic device comprisingat least one sample collection unit, an assay assembly; and a pluralityof channels in fluid communication with said sample collection unitand/or said assay assembly; b) allowing a sample of biological fluid ofless than about 50 ul to react with reactants contained within saidassay assembly to yield a detectable signal generated from an analyteinitially collected in said sample that is indicative of apharmacological parameter; and c) detecting said detectable signal; andd) repeating the reaction and detection steps with a second sample ofbiological fluid from the same test animal. In yet another embodiment,the method utilizes test animals that are not subjected to anesthesia.

The present invention provides a method of improving the accuracy ofcalibrating a fluidic system, comprising: a) providing a system fordetecting an analyte in a bodily fluid from a subject comprising afluidic device for providing said bodily fluid, said fluidic devicehaving a calibration assembly and a reader assembly for detecting thepresence of said analyte; b) measuring one or more parameters of acalibration curve associated with said fluidic device; c) comparing saidone or more parameters with predetermined parameters associate with saidfluidic device; d) adjusting a signal output by the ratio of said one ormore parameters and said predetermined parameters. The present inventionalso provides a method of improving the calibration of a fluidic system.The method involves the steps of a) measuring a first signal in anoriginal sample comprising a known quantity of an analyte; b) measuringa second signal after spiking said original sample with a known quantityof said analyte; c) plotting the difference between said first andsecond signals against a target value, wherein said target value is asignal expected for said known quantity of said analyte; and d) arrivingat a best fit of parameters by minimizing the sum of the square of thedifferences between said target value and calculated analyte values.

Further provided by the present invention is a method of assessing thereliability of an assay for an analyte in a bodily fluid with the use ofa fluidic device, comprising: a) providing a system, said systemcomprising a fluidic device, said fluidic device comprising a samplecollection unit and an assay assembly, wherein said sample collectionunit allows a sample of bodily fluid to react with reactants containedwithin said assay assembly, for detecting the presence of an analyte ina bodily fluid from a subject, and a reader assembly for detecting thepresence of said analyte; b) sensing with a sensor a change in operationparameters under which the system normally operates

The present invention also provides a method of performing a trendanalysis on the concentration of an analyte in a subject. The methodinvolves the steps of a) providing a fluidic device comprising at leastone sample collection unit, an immunoassay assembly containingimmunoassay reagents, a plurality of channels in fluid communicationwith said sample collection unit and/or said immunoassay assembly; b)actuating said fluidic device and directing said immunoassay reagentswithin said fluidic device; c) allowing a sample of bodily fluid of lessthan about 500 ul to react with said immunoassay reagents containedwithin said assay immunoassay assembly to yield a detectable signalindicative of the presence of said analyte in said sample; d) detectingsaid detectable signal generated from said analyte collected in saidsample of bodily fluid; and e) repeating steps a) through d) for asingle patient over a period of time to detect concentrations of saidanayte, thereby performing said trend analysis.

The present invention provides an apparatus for detecting an analyte ina biological fluid of a subject, wherein a plurality of reaction sitescomprises an optical barrier. In one aspect, the bound reactants in atleast one reaction site are unevenly distributed, for example beinglocalized around the center of said reaction site. The present inventionalso provides a method of using such apparatus.

Finally, the present invention provides a method of manufacturing afluidic device for detecting an analyte in a biological fluid of asubject. The method involves the steps of a) providing a plurality oflayers of a fluidic device; b) ultrasonically welding said layerstogether such that a fluidic network exists between a sample collectionunit, at least one reactant chamber, at least one reaction site, and atleast one waste chamber.

In practice the subject invention, the reactants contained in thedevices may comprise immunoassay reagents. In one aspect, theimmunoassay reagents detect a microorganism selected from the groupconsisting of bacterium, virus, fungus, and protozoa. In another aspect,the immunoassay reagents may detect a polypeptide glycoprotein,polysaccharide, lipid, nucleic acid, and a combination thereof. Inanother aspect, the immunoassay reagents detect a member selected fromthe group consisting of drug, drug metabolite, biomarker indicative of adisease, tissue specific marker, and biomarker specific for a cell orcell type. In yet another aspect, the immunoassay generates luminescentsignals, preferably chemiluminescent signals. Where desired, the subjectfluidic device can be configured to detect a plurality of analytes. Theplurality of analytes can be identified by distinct signals detectableover a range of 3 orders of magnitude. The detectable signal can be aluminescent signal, including but not limited to photoluminescence,electroluminescence, chemiluminescence, fluorescence, phosphorescence.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is one embodiment showing multiple components of the presentsystem.

FIG. 2 shows different layers of an exemplary fluidic device prior toassembly.

FIGS. 3 and 4 illustrate the fluidic network within an exemplary fluidicdevice.

FIG. 5 shows a top, side, and bottom view of exemplary reagent chambersof the present invention.

FIG. 6 illustrates an exemplary side view of a reagent chamber influidic communication with a fluidic device.

FIG. 7 illustrates exemplary reagent chambers being filled withreagents.

FIGS. 8 and 9 illustrate a side view of an exemplary fluidic device iscombination with actuating elements of the reader assembly.

FIG. 10 compares a two-step assay with a competitive binding assay.

FIG. 11 shows an exemplary two-step chemiluminescence enzymeimmunoassay.

FIG. 12 shows the increased sensitivity of the two-stepchemiluminescence enzyme immunoassay.

FIG. 13 shows the ability of TOSCA to assay less than ideal samples andmaintain desired sensitivity.

FIGS. 14A-C illustrate exemplary fluidic channels between reactionsites.

FIGS. 15A and 15B illustrate reactions sites to reduce the signal fromunbound conjugates remaining in reaction sites.

FIG. 16 shows an exemplary bubble trapper or remover to prevent bubblesfrom entering the reaction sites.

FIG. 17 shows the sensitivity enhancement achieved using TOSCA ascompared with competitive binding.

FIG. 18 shows two analytes, prostacyclin metabolite and thromboxanemetabolite, which have been identified and quantified and theirconcentrations are different by more than 3 orders of magnitude.

FIG. 19 shows an exemplary flow chart of a business method of monitoringa clinical trial of a therapeutic agent.

FIG. 20 shows simultaneous sharing of the information detected with afluidic device with various interested parties.

FIG. 21 shows a typical assay dose-response data for a two-step assayfor TxB2.

FIG. 22 shows dose responses computed with and without errors incalibration parameters.

FIG. 23 shows computed concentration errors produced by 1%mis-estimation of A and D calibration values.

FIG. 24 illustrates calibration using a “differential” approach.

FIG. 25 shows the verification of calibration using the “1-point spike”method (log scale).

FIG. 26 shows the verification of calibration using the “1-point spike”method (linear scale).

FIG. 27 shows dose-response of assays calibrated against a plasma samplewith a very low TxB2 concentration.

FIG. 28 shows use of spike recovery to eliminate calibration errors ofthe “C” parameter.

FIG. 29 illustrates calculating differences in concentration between twosamples.

FIG. 30 illustrates an assay of plasma samples.

FIG. 31 shows the time course of assay signal generation.

FIG. 32 shows the impact of change in calibration parameter “A” on assaycalibration.

FIG. 33 shows how a reference therapeutic index would be computed.

FIG. 34 illustrates computing the therapeutic index.

FIG. 35 shows multiple regression analysis of the computed therapeuticindex.

FIG. 36 is an illustration of the relationship between measured drug,analyte and biomarker concentration and therapeutic index.

FIG. 37 is an illustration of the application of this invention tominimize adverse drug reactions.

FIG. 38 shows exemplary patient input values.

FIG. 39 shows use of a therapeutic index to follow treatment progressionin an autism patient.

DETAILED DESCRIPTION OF THE INVENTION System

One aspect of the present invention is a system for detecting an analytein a sample of bodily fluid. The system is capable of detecting and/orquantifying analytes that are associated with specific biologicalprocesses, physiological conditions, disorders or stages of disorders.

The subject system comprises a fluidic device having one or more of thefollowing components: a sample collection unit, an assay assembly, areader assembly, and a communication assembly. The sample collectionunit typically allows a sample of bodily fluid collected from a subjectto react with reactants contained within the assay assembly forgenerating a signal indicative of the presence of the analyte ofinterest. The reader assembly detects the signal, which is thentransmitted via the communication assembly to an external device forfurther processing.

Any bodily fluids suspected to contain an analyte of interest can beused in conjunction with the subject system or devices. Commonlyemployed bodily fluids include but are not limited to blood, serum,saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginalfluid, interstitial fluids derived from tumorous tissue, andcerebrospinal fluid. In a preferred embodiment, the bodily fluids areused directly for detecting the analytes present therein with thesubject fluidic device without further processing. Where desired,however, the bodily fluids can be pre-treated before performing theanalysis with the subject fluidic devices. The choice of pre-treatmentswill depend on the type of bodily fluid used and/or the nature of theanalyte under investigation. For instance, where the analyte is presentat low level in a sample of bodily fluid, the sample can be concentratedvia any conventional means to enrich the analyte. Methods ofconcentrating an analyte include but are not limited to drying,evaporation, centrifugation, sedimentation, precipitation, andamplification. Where the analyte is a nucleic acid, it can be extractedusing various lytic enzymes or chemical solutions according to theprocedures set forth in Sambrook et al. (“Molecular Cloning: ALaboratory Manual”), or using nucleic acid binding resins following theaccompanying instructions provided by manufactures. Where the analyte isa molecule present on or within a cell, extraction can be performedusing lysing agents including but not limited to denaturing detergentsuch as SDS or non-denaturing detergent such as thesit, sodiumdeoxylate, triton X-100, and tween-20.

The volume of bodily fluid to be used with a fluidic device of thepresent invention is generally less than about 500 microliters,typically between about 1 to 100 microliters. Where desired, a sample of1 to 50 microliters or 1 to 10 microliters can be used for detecting ananalyte using the subject fluidic device.

A bodily fluid may be drawn from a patient and brought into the fluidicdevice in a variety of ways, including but not limited to, lancing,injection, or pipetting. In one embodiment, a lancet punctures the skinand draws the sample into the fluidic device using, for example,gravity, capillary action, aspiration, or vacuum force. The lancet maybe part of the fluidic device, or part of a reader assembly, or as astand alone component. Where needed, the lancet may be activated by avariety of mechanical, electrical, electromechanical, or any other knownactivation mechanism or any combination of such methods. In anotherembodiment where no active mechanism is required, a patient can simplyprovide a bodily fluid to the fluidic device, as for example, couldoccur with a saliva sample. The collected fluid can be placed in thesample collection unit within the fluidic device. In yet anotherembodiment, the fluidic device comprises at least one microneedle whichpunctures the skin. The microneedle can be used with a fluidic devicealone, or can puncture the skin after the fluidic device is insertedinto a reader assembly.

In some embodiments a microneedle is about the size of a human hair andhas an integrated microreservoir or cuvette. The microneedle maypainlessly penetrate the skin and draw a small blood sample. Morepreferably, the microneedle collects about 0.01 to about 1 microliter,preferably about 0.05 to about 0.5 microliters and more preferably about0.1 to about 0.3 microliters of capillary blood. In some embodiments amicroneedle may be constructed out of silicon and is about 10 to about200 microns in diameter, preferably about 50 to about 150 microns indiameter, and most preferably about 100 microns in diameter, makingtheir application to the skin virtually painless. To ensure that acapillary is actually struck by a needle, a plurality of microneedlesmay be used for sample collection. Such microneedles may be of the typemarketed by Pelikan (Palo Alto, Calif.) and/or Kumetrix (Union City,Calif.). U.S. Pat. No. 6,503,231 discloses microneedles which may beused with the present invention.

Microfabrication processes that may be used in making the microneedlesdisclosed herein include without limitation lithography; etchingtechniques such as wet chemical, dry, and photoresist removal; thermaloxidation of silicon; electroplating and electroless plating; diffusionprocesses such as boron, phosphorus, arsenic, and antimony diffusion;ion implantation; film deposition such as evaporation (filament,electron beam, flash, and shadowing and step coverage), sputtering,chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, andmolecular beam), electroplating, screen printing, and lamination. Seegenerally Jaeger, Introduction to Microelectronic Fabrication(Addison-Wesley Publishing Co., Reading Mass. 1988); Runyan, et al.,Semiconductor Integrated Circuit Processing Technology (Addison-WesleyPublishing Co., Reading Mass. 1990); Proceedings of the IEEE MicroElectro Mechanical Systems Conference 1987-1998; Rai-Choudhury, ed.,Handbook of Microlithography, Micromachining & Microfabrication (SPIEOptical Engineering Press, Bellingham, Wash. 1997). Alternatively,microneedles may be molded in silicon wafers and then plated usingconventional wire cutting techniques with nickel, gold, titanium orvarious other biocompatible metals. In some embodiments microneedles canbe fashioned from biopolymers. In some embodiments microneedles may befabricated and employed for the claimed devices according to the methodsof Mukerjee et al., Sensors and Actuators A: Physical, Volume 114,Issues 2-3, 1 Sep. 2004, Pages 267-275.

In preferred embodiments a microneedle is only used once and thendiscarded. In some embodiments a mechanical actuator can insert andwithdraw the microneedle from the patient, discard the used needle, andreload a new microneedle. The mechanical technologies developed andmanufactured in very high volumes for very small disk drives have asimilar set of motion and low cost requirements. In preferredembodiments the actuator is a MEMS (micro machined electromechanicalsystem) device fabricated using semiconductor-like batch processes. Suchactuators include without limitation nickel titanium alloy, neumatic, orpiezo electric devices. In some embodiments the microneedles are about 1micron to about 10 microns in thickness, preferably about 2 microns toabout 6 microns in thickness, and most preferably about 4 microns inthickness. In some embodiments the microneedles are about 10 microns toabout 100 microns in height, preferably about 30 microns to about 60microns in height, and most preferably about 40 microns in height.

FIG. 1 illustrates an exemplary system of the present invention. Asillustrated, a fluidic device provides a bodily fluid from a patient andcan be inserted into a reader assembly. The fluidic device may take avariety of configurations and in some embodiments the fluidic device maybe in the form of a cartridge. An identifier (ID) detector may detect anidentifier on the fluidic device. The identifier detector communicateswith a communication assembly via a controller which transmits theidentifier to an external device. Where desired, the external devicesends a protocol stored on the external device to the communicationassembly based on the identifier. The protocol to be run on the fluidicdevice may comprise instructions to the controller of the readerassembly to perform the protocol on the fluidic device, including butnot limited to a particular assay to be run and a detection method to beperformed. Once the assay is performed on the fluidic device, a signalindicative of an analyte in the bodily fluid sample is generated anddetected by a detection assembly. The detected signal may then becommunicated to the communications assembly, where it can be transmittedto the external device for processing, including without limitation,calculation of the analyte concentration in the sample.

FIG. 2 illustrates exemplary layers of a fluidic device according to thepresent invention prior to assembly of the fluidic device which isdisclosed in more detail below. FIGS. 3 and 4 show a top and bottomview, respectively, of an exemplary fluidic device after the device hasbeen assembled. The different layers are designed and assembled to forma three dimensional fluidic channel network. A sample collection unit 4provides a sample of bodily fluid from a patient. As will be explainedin further detail below a reader assembly comprises actuating elements(not shown) can actuate the fluidic device to start and direct the flowof a bodily fluid sample and assay reagents in the fluidic device. Insome embodiments actuating elements first cause the flow of sample inthe fluidic device 2 from sample collection unit 4 to reaction sites 6,move the sample upward in the fluidic device from point G′ to point G,and then to waste chamber 8. The actuating elements then initiate theflow of reagents from reagent chambers 10 to point B′, point C′, andpoint D′, then upward to points B, C, and D, respectively, then to pointA, down to point A′, and then to waste chamber 8 in the same manner asthe sample.

A sample collection unit 4 in a fluidic device 2 may provide a bodilyfluid sample from a patient by any of the methods described above. Ifnecessary, the sample may first be processed by diluting the bodilyfluid in a dilution chamber, and or may be filtered by separating theplasma from the red blood cells in a filtration chamber. In someembodiments the sample collection unit, diluting chamber, and filtrationchamber may be the same component, and in some embodiments they may bedifferent components, or any two may be the same component and the othermay be a separate component. In some embodiments there may be more thanone sample collection unit in the fluidic device.

In some embodiments it may be desirable to detect the presence ofanalytes on a cell surface, within a cell membrane, or inside a cell.The difficulty of detecting such analytes is that cells and other formedelements are particulate and components of cells do not readily interactwith traditional assay chemistries which are designed to operate onanalytes in solution. Cell-surface analytes react slowly andinefficiently with surface bound probes, and analytes inside the cellcan not react at all with bound probes. To allow the detection of suchanalytes, in some embodiments the fluidic device may include a lysingassembly to lyse cells present in the bodily fluid sample. The lysingassembly may be incorporated with the sample collection unit, a dilutionchamber, and/or a filtration chamber. In some embodiments the samplecollection unit, dilution chamber, and lysing component are within thesame element in the fluidic device. In some embodiments the lysingcomponent may be incorporated with an assay reagent described below.

Where desired, lysing agents may be impregnated and then dried intoporous mats, glass fiber mats, sintered fits or particles such as Porex,paper, or other similar material. Lysing agents may be dried onto flatsurfaces. Lysing agents may also be dissolved in liquid diluents orother liquid reagents. In preferred embodiments porous materials areused to store the lysing agents because they can store a lysing agent indry form likely to be very stable. They also facilitate the mixing ofthe bodily fluid sample with the lysing agent by providing a tortuouspath for the sample as it moves through the porous material. Inpreferred embodiments such porous materials have a disc shape with adiameter greater than its thickness. In some embodiments lysing agentsmay be dried onto porous materials using lyophilization, passiveevaporation, exposure to warm dry flowing gas, or other known methods.

A variety of lysing agents are available in the art and are suitable foruse in connection with the subject fluidic device. Preferred lysingagents are non-denaturing, such as non-denaturing detergents.Non-limiting examples of non-denaturing detergents include thesit,sodium deoxylate, triton X-100, and tween-20. The agents are preferablynon-volatile in embodiments where the agents are impregnated into asolid porous materials. In some embodiments lysing agents are mixedtogether. Other materials may be mixed with the lysing agents to modifythe lytic effects. Such exemplary materials may be, without limitation,buffers, salts, and proteins. In preferred embodiments lysing agentswill be used in amounts that are in excess of the minimum amountrequired to lyse cells. In some embodiments lysing agents will be usedthat can lyse both white and red cells.

One of the advantages of the present invention is that any reagentsnecessary to perform an assay on a fluidic device according to thepresent invention are preferably on-board, or housed within the fluidicdevice before, during, and after the assay. In this way the only inletor outlet from the fluidic device is preferably the bodily fluid sampleinitially provided by the fluidic device. This design also helps createan easily disposable fluidic device where all fluids or liquids remainin the device. The on-board design also prevents leakage from thefluidic device into the reader assembly which should remain free fromcontamination from the fluidic device.

In a preferred embodiment there is at least one reagent chamber. In someembodiments there may be two, three, four, five, six, or more, or anynumber of reagent chambers as are necessary to fulfill the purposes ofthe invention. A reagent chamber is preferably in fluid communicationwith at least one reaction site, and when the fluidic device is actuatedas described herein, reagents contained in said reagent chambers arereleased into the fluidic channels within the fluidic device.

Reagents according to the present invention include without limitationwash buffers, enzyme substrates, dilution buffers, conjugates,enzyme-labeled conjugates, DNA amplifiers, sample diluents, washsolutions, sample pre-treatment reagents including additives such asdetergents, polymers, chelating agents, albumin-binding reagents, enzymeinhibitors, enzymes, anticoagulants, red-cell agglutinating agents,antibodies, or other materials necessary to run an assay on a fluidicdevice. An enzyme conjugate can be either a polyclonal antibody ormonoclonal antibody labeled with an enzyme that can yield a detectablesignal upon reaction with an appropriate substrate. Non-limitingexamples of such enzymes are alkaline phosphatase and horseradishperoxidase. In some embodiments the reagents comprise immunoassayreagents.

In some embodiments a reagent chamber contains approximately about 50 μlto about 1 ml of fluid. In some embodiments the chamber may containabout 100 μl of fluid. The volume of liquid in a reagent chamber mayvary depending on the type of assay being run or the sample of bodilyfluid provided. In some embodiments the reagents are initially storeddry and liquified upon initiation of the assay being run on the fluidicdevice.

FIGS. 5 and 6 illustrate an exemplary embodiment of a sealed reagentchamber. FIG. 5 shows a top, side, and bottom view of a reagent chamber.A top layer 11 contains a plurality of bubbles or pouches 13. A bottomlayer 15 has a bottom surface that is bonded to the fluidic device base17 as shown in FIG. 6. The bottom layer 15 has a plurality of fluidicchannels 19 dispersed through the entire surface, where each channeltraverses the bottom layer 15. The fluid in the reagent chamber iscontained within the chamber by pressure burstable seal 21 between thefluidic channel 19 and the chamber 13. The burstable seal 21 is designedsuch that at a pre-determined pressure the seal bursts allowing thefluid in the chamber 13 to flow out into a fluidic channel 19.

FIG. 7 shows an exemplary process of filling the reagent chambers 13with, for example, reagents. Reagent chambers 13 may be filled withfluid using a fill channel and a vacuum draw channel. The process offilling the reagents involves first removing all the air from thechamber. This is done by drawing a vacuum through the vacuum drawchannel. Once the vacuum is drawn, a permanent seal is placed betweenthe fill channel and the vacuum draw channel. Next, required reagentsare dispensed into the chamber through the fill channel. Then, apermanent seal is placed between the chamber and the fill channel. Thisensures that when the chamber is compressed, the fluid can flow in onlyone direction, towards the burstable seal. If the compression imparts apressure larger than the burst pressure of seal, the seal bursts and thefluid flows into the fluidic channel.

FIGS. 8 and 9 illustrate an embodiment of a fluidic device in operationwith actuating elements as described herein. Fluidic device 2 contains areagent chamber 10 and a layer of burstable foil 12 enclosing thereagent chamber. Above the burstable foil 12 is a portion of themicrofluidic circuit 14. A tough, but elastomeric top cover 16 acts asthe top layer of the fluidic device 2. The reader assembly includes avalve actuation plate 18. Securely attached to the plate 18 is anon-coring needle 20 such that when the plate is lowered, the sharp edgeof the needle contacts the elastomeric cover 16. The top cover couldalso be made of flexible silicone material that would act as a moistureimpermeable seal. This embodiment also provides a solution to liquidevaporation and leakage from a fluidic device by isolating any liquidreagents in the fluidic device from any dry reagents until the assay isinitiated.

In preferred embodiments the reagent chamber and sample collection unitare fluidly connected to reaction sites where bound probes can detect ananalyte of interest in the bodily fluid sample using the assay. Areaction site could then provide a signal indicative of the presence ofthe analyte of interest, which can then be detected by a detectiondevice described in detail herein below.

In some embodiments the reactions sites are flat but they may take on avariety of alternative surface configurations. The reaction sitepreferably forms a rigid support on which a reactant can be immobilized.The reaction site surface is also chosen to provide appropriatelight-absorbing characteristics. For instance, the reaction site may befunctionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, polypropylene, or combinations thereof. Other appropriatematerials may be used in accordance with the present invention.

A reactant immobilized at a reaction site can be anything useful fordetecting an analyte of interest in a sample of bodily fluid. Forinstance, such reactants include without limitation nucleic acid probes,antibodies, cell membrane receptors, monoclonal antibodies and antiserareactive with a specific analyte. Various commercially availablereactants such as a host of polyclonal and monoclonal antibodiesspecifically developed for specific analytes can be used.

One skilled in the art will appreciate that there are many ways ofimmobilizing various reactants onto a support where reaction can takeplace. The immobilization may be covalent or noncovalent, via a linkermoiety, or tethering them to an immobilized moiety. These methods arewell known in the field of solid phase synthesis and micro-arrays (Beieret al., Nucleic Acids Res. 27:1970-1-977 (1999). Non-limiting exemplarybinding moieties for attaching either nucleic acids or proteinaceousmolecules such as antibodies to a solid support include streptavidin oravidin/biotin linkages, carbamate linkages, ester linkages, amide,thiolester, (N)-functionalized thiourea, functionalized maleimide,amino, disulfide, amide, hydrazone linkages, and among others. Inaddition, a silyl moiety can be attached to a nucleic acid directly to asubstrate such as glass using methods known in the art.

In some embodiments there are more than one reaction sites which canallow for detection of multiple analytes of interest from the samesample of bodily fluid. In some embodiments there are 2, 3, 4, 5, 6, ormore reaction sites, or any other number of reaction sites as may benecessary to carry out the intent of the invention.

In embodiments with multiple reaction sites on a fluidic device, eachreaction site may be immobilized with a reactant different from areactant on a different reaction site. In a fluidic device with, forexample, three reaction sites, there may be three different probes, eachbound to a different reaction site to bind to three different analytesof interest in the sample. In some embodiments there may be differentreactants bound to a single reaction site if, for example, a CCD withmultiple detection areas were used as the detection device, such thatmultiple different analytes could be detected in a single reaction site.The capability to use multiple reaction sites in addition to multipledifferent probes on each reaction site enables the high-throughputcharacteristics of the present invention.

The present invention allows for the detection of multiple analytes onthe same fluidic device. If assays with different luminescentintensities are run in adjacent reaction sites, photons (signals thatemanate from the reactions) may travel from one reaction site to anadjacent reaction site, as reaction sites may be constructed ofmaterials that allow photons to travel through the fluidic channels thatconnect the sites. This optical cross talk may compromise the accuracyof the detected photons. FIGS. 14B and 14C illustrate differentembodiments of this invention that can eliminate or reduce the amount ofoptical cross-talk. Non-linear channels 22 will not allow photons(light) to pass through. Hence, embodiments such as those shown in FIGS.14B and 14C would not allow signals from a reaction site to contaminatea signal produced from an adjacent site from which a detection devicemay be detecting. Additionally, the edges or walls of a reaction sitemay be constructed using optically opaque materials so that light willnot escape the wells. In some embodiments the reaction sites are whiteor opaque.

In some embodiments, unbound conjugates may need to be washed from areaction site to prevent unbound conjugates from activating thesubstrate and producing and inaccurate signal. It may be difficult toremove conjugates sticking to the edges of the reaction sites in such afluidic device if, for example, there is not an excess of a washsolution. To decrease the signal contributed from unbound conjugatesstuck to the edge of a reaction site, it may be advantageous to expandthe reaction site edge or wall radius in order to distance stuckconjugate from the desired actual detection area, represented by boundreactant. FIGS. 15A and 15B illustrates this concept. Reaction site 6contains reaction surface 24 and edge or wall surface 26. In FIG. 15B,an edge surface 26 is shown at a greater distance from the center of thereaction site 6 than is the edge surface of the prior art design. Thisallows unbound conjugates to adhere to the edge surfaces and bedistanced from bound conjugates, which are concentrated closer to thecenter of the reaction site 6.

In preferred embodiments of the invention the fluidic device includes atleast one waste chamber to trap or capture all liquids after they havebeen used in the assay. In preferred embodiments, there is more than onewaste chamber, at least one of which is to be used with a calibrationassembly described herein below. On-board waste chambers also allow thedevice to be easily disposable. The waste chamber is preferably influidic communication with at least one reaction site.

At least one of these channels will typically have small cross sectionaldimensions. In some embodiments the dimensions are from about 0.01 mm toabout 5 mm, preferably from about 0.03 mm to about 3 mm, and morepreferably from about 0.05 mm to about 2 mm. Fluidic channels in thefluidic device may be created by, for example without limitation,precision injection molding, laser etching, or any other technique knownin the art to carry out the intent of the invention.

One of the common problems encountered in a microfluidic based assaysystem is the presence of air or gas bubbles. It is extremely difficultto remove a bubble once it is trapped within a fluidic channel. Bubblespresent anywhere in the fluidic circuit, particularly in the reactionsites can compromise the assay capabilities. A bubble may end upoccupying part of all of the surface area of a reaction site.Consequently the reader may end up reading a muted signal or no signalat all. FIG. 16 illustrates an embodiment where a bubble could betrapped in a filter 28 before it reaches a reaction site 6. A bubbletrapper 28 can be positioned between a sample collection unit 4 andreaction site 6. The bubble trapper can have such a geometry that thebubbles tend to migrate towards the edges of this surface and remainstuck at that service, thereby not entering into the reaction sites.

To ensure that a given photon count produced at a reaction sitecorrelates with an accurate concentration of an analyte of interest in asample, it is preferably advantageous to calibrate the fluidic devicebefore detecting the photons. Calibrating a fluidic device at the pointof manufacturing for example may be insufficient to ensure an accurateanalyte concentration is determined because a fluidic device may beshipped prior to use and may undergo changes in temperature, forexample, so that a calibration performed at manufacturing does not takeinto effect any subsequent changes to the structure of the fluidicdevice or reagents contained therein. In a preferred embodiment of thepresent invention, a fluidic device has a calibration assembly thatmimics the assay assembly in components and design except that a sampleis not introduced into the calibration assembly. Referring to FIGS. 3and 4, a calibration assembly occupies about half of the fluidic device2 and includes reagent chambers 32, reactions sites 34, a waste chamber36, and fluidic channels 38. Similar to the assay assembly, the numberof reagent chambers and reaction sites may vary depending on the assaybeing run on the fluidic device and the number of analytes beingdetected.

Where desired, a sensor for assessing the reliability of an assay for ananalyte in a bodily fluid with the use of the subject fluidic device canbe provided together with the fluidic device, the reader and/or withinthe packaging of the subject system. The sensor is capable of detectinga change in operation parameters under which the subject system normallyoperates. The operation parameters include but are not limited totemperature, humidity, and pressure, which may affect the performance ofthe present system.

A fluidic device and reader assembly may, after manufacturing, beshipped to the end user, together or individually. As a reader assemblyis repeatedly used with multiple fluidic devices, it may be necessary tohave sensors on both the fluidic device and reader assembly to detectsuch changes during shipping, for example. During shipping, pressure ortemperature changes can impact the performance of a number of componentsof the present system, and as such a sensor located on either thefluidic device or reader assembly can relay these changes to, forexample, the external device so that adjustments can be made duringcalibration or during data processing on the external device. Forexample, if the pressure of a fluidic device dropped to a certain levelduring shipping, a sensor located on the fluidic device could detectthis change and convey this information to the reader assembly when itis inserted into the reader assembly by the user. There may be anadditional detection device in the reader assembly to perform this, orsuch a device may be incorporated into another system component. In someembodiments this information may be wirelessly transmitted to either thereader assembly or the external device. Likewise, a sensor in the readerassembly can detect similar changes. In some embodiments, it may bedesirable to have a sensor in the shipping packaging as well, eitherinstead of in the system components or in addition thereto.

Manufacturing of the fluidic channels may generally be carried out byany number of microfabrication techniques that are well known in theart. For example, lithographic techniques are optionally employed infabricating, for example, glass, quartz or silicon substrates, usingmethods well known in the semiconductor manufacturing industries such asphotolithographic etching, plasma etching or wet chemical etching.Alternatively, micromachining methods such as laser drilling,micromilling and the like are optionally employed. Similarly, forpolymeric substrates, well known manufacturing techniques may also beused. These techniques include injection molding or stamp moldingmethods where large numbers of substrates are optionally produced using,for example, rolling stamps to produce large sheets of microscalesubstrates or polymer microcasting techniques where the substrate ispolymerized within a micromachined mold.

In some embodiments at least one of the different layers of the fluidicdevice may be constructed of polymeric substrates. Non limiting examplesof polymeric materials include polystyrene, polycarbonate,polypropylene, polydimethysiloxanes (PDMS), polyurethane,polyvinylchloride (PVC), and polysulfone.

The fluidic device may be manufactured by stamping, thermal bonding,adhesives or, in the case of certain substrates, for example, glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. In some embodiments the fluidic device ismanufactured by ultrasonic or acoustic welding.

FIG. 2 shows one embodiment of the invention in which fluidic device 2is comprised of 7 layers. Features as shown are, for example, cut in thepolymeric substrate such that when the layers are properly positionedwhen assembly will form a fluidic network. In some embodiments more orfewer layers may be used to construct a fluidic device to carry out thepurpose of the invention.

One objective of the present invention is to prevent fluid inside afluidic device from contacting the components of a reader assembly whichmay need to remain dry and or uncontaminated, and also to preventcontamination to a detection device within the reader assembly. A leakin the fluidic device could result in liquids, for example reagents orwaste, escaping from the fluidic device and contaminating the reader. Inother embodiments a liquid absorbing material, such as polymericmaterials found in diapers, could be placed within a portion of thefluidic channel or waste chamber to absorb the waste liquid. Anon-limiting example of such a polymer is sodium polyacrylate. Suchpolymers can absorb fluids hundreds of times their weight. Hence, onlyminute quantities of such polymeric materials may be required toaccomplish the goal of absorbing leaked fluids. In some embodiments awaste chamber is filled with a superabsorbent material. In someembodiments leaked liquid may be converted into a gel or other solid orsemi-solid form.

Another objective of the present system is to provide a fluidic devicethat can run a variety of assays on a fluidic device, regardless of theanalyte being detected from a bodily fluid sample. A protocol dependenton the identity of the fluidic device may be transferred from anexternal device where it can be stored to a reader assembly to enablethe reader assembly to carry out the specific protocol on the fluidicdevice. In preferred embodiments, the fluidic device has an identifier(ID) that is detected or read by an identifier detector describedherein. The identifier can then be communicated to a communicationassembly, where it can then be transferred or transmitted to an externaldevice.

In some embodiments the identifier may be a bar code identifier with aseries of black and white lines, which can be read by an identifierdetector such as a bar code reader, which are well known. Otheridentifiers could be a series of alphanumerical values, colors, raisedbumps, or any other identifier which can be located on a fluidic deviceand be detected or read by an identifier detector. In some embodimentsthe identifier may comprise a storage or memory device and can transmitinformation to an identification detector. In some embodiments bothtechniques may be used.

Once a bodily fluid sample is provided to a fluidic device, it isinserted in a reader assembly. In some embodiments the fluidic device ispartially inserted manually, and then a mechanical switch in the readerassembly automatically properly positions the fluidic device inside thereader assembly. Any other mechanism known in the art for inserting adisk or cartridge into a device may be used as well. In some embodimentsonly manual insertion may be required.

In some embodiments the reader assembly comprises an identifier detectorfor detecting or reading an identifier on the fluidic device, acontroller for automatically controlling the detection assembly and alsomechanical components of the reader assembly, for example, pumps and/orvalves for controlling or directing fluid through the fluidic device, adetection device for detecting a signal created by an assay run on thefluidic device, and a communication assembly for communicating with anexternal device.

An identifier detector detects an identifier on the fluidic device whichis communicated to a communication assembly. In some embodiments theidentifier detector can be a bar code scanner-like device, reading a barcode on a fluidic device. The identifier detector may also be an LEDthat emits light which can interact with an identifier which reflectslight and is measured by the identifier detector to determine theidentity of a fluidic device.

In preferred embodiments the reader assembly houses a controller whichcontrols a pump and a series of valves to control and direct the flow ofliquid within the fluidic device. In some embodiments the readerassembly may comprises multiple pumps. The sample and reagents arepreferably pulled through the fluidic channels by a vacuum force createdby sequentially opening and closing at least one valve while activatinga pump within the reader assembly. Methods of using at least one valveand at least one pump to create a vacuum force are well known. While anegative pulling force may be used, a positive pushing force may also begenerated by at least one pump and valve according to the presentinvention. In other embodiments movement of fluid on the fluidic devicemay be by electro-osmotic, capillary, piezoelectric, or microactuatoraction.

FIGS. 8 and 9 illustrate an exemplary sequence to initiate the flow of areagent within the fluidic device. An actuation plate 18 in the readerassembly comprises a non-coring needle or pin 20 which when loweredflexes the top cover 16, as it is preferably made of strong, flexibleelastomeric material. However, the easily rupturable foil 12 thenruptures due to the stress induced by the flexing of top cover 16.Valves located downstream to the reagent chamber puncture differentareas of foil in the fluidic device and can then work in tandem with apump within the reader assembly to create a vacuum force to pull thereagent out of the reagent chamber 6 into a fluidic channel and thendirect the flow of the reagent to a reaction site. At least one valve ispreferably fluidically connected to a pump housed within the readerassembly. The non-coring needle or pin 20 is removed from the fluidicdevice when the device is removed from the reader assembly. One of theadvantages of this embodiment is that no on-chip pump is required,which, at least, decreases the size and cost of the fluidic device, andallows the device to be disposable.

A reaction assembly preferably houses a detection assembly for detectinga signal produced by at least one assay on the fluidic device. FIG. 1illustrates an exemplary position of a detection device of the presentinvention in relation to the fluidic device which is below the fluidicdevice. The detection assembly may be above the fluidic device or at adifferent orientation in relation to the fluidic device based on, forexample, the type of assay being performed and the detection mechanismbeing employed.

In preferred embodiments an optical detector is used as the detectiondevice. Non-limiting examples include a photodiode, photomultiplier tube(PMT), photon counting detector, or charge-coupled device (CCD). In someembodiments a pin diode may be used. In some embodiments a pin diode canbe coupled to an amplifier to create a detection device with asensitivity comperable to a PMT. Some assays may generate luminescenceas described herein. In some embodiments chemiluminescence is detected.In some embodiments a detection assembly could include a plurality offiber optic cables connected as a bundle to a CCD detector or to a PMTarray. The fiber optic bundle could be constructed of discrete fibers orof many small fibers fused together to form a solid bundle. Such solidbundles are commercially available and easily interfaced to CCDdetectors.

In some embodiments, the detection system may comprise non-opticaldetectors or sensors for detecting a particular parameter of a patient.Such sensors may include temperature, conductivity, potentiometric, andamperometric, for compounds that are oxidized or reduced, for example,O₂, H₂O₂, and I₂, or oxidizable/reducible organic compounds.

A communication assembly is preferably housed within the reader assemblyand is capable of transmitting and receiving information wirelessly froman external device. Such wireless communication may be bluetooth or RTMtechnology. Various communication methods can be utilized, such as adial-up wired connection with a modem, a direct link such as a TI, ISDN,or cable line. In preferred embodiments a wireless connection isestablished using exemplary wireless networks such as cellular,satellite, or pager networks, GPRS, or a local data transport systemsuch as Ethernet or token ring over a local area network. In someembodiments the information is encrypted before it is transmitted over awireless network. In some embodiments the communication assembly maycontain a wireless infrared communication component for sending andreceiving information.

In some embodiments the communication assembly can have a memory orstorage device, for example localized RAM, in which the informationcollected can be stored. A storage device may be required if informationcan not be transmitted at a given time due to, for example, a temporaryinability to wirelessly connect to a network. The information can beassociated with the fluidic device identifier in the storage device. Insome embodiments the communication assembly can retry sending the storedinformation after a certain amount of time. In some embodiments thememory device can store the information for a period of ten days beforeit is erased.

In preferred embodiments an external device communicates with thecommunication assembly within the readers assembly. An external devicecan wirelessly communicate with a reader assembly, but can alsocommunicate with a third party, including without limitation a patient,medical personnel, clinicians, laboratory personnel, or others in thehealth care industry.

In some embodiments the external device can be a computer system,server, or other electronic device capable of storing information orprocessing information. In some embodiments the external device includesone or more computer systems, servers, or other electronic devicescapable of storing information or processing information. In someembodiments an external device may include a database of patientinformation, for example but not limited to, medical records or patienthistory, clinical trial records, or preclinical trial records. Inpreferred embodiments, an external device stores protocols to be run ona fluidic device which can be transmitted to the communication assemblyof a reader assembly when it has received an identifier indicating whichfluidic device has been inserted in the reader assembly. In someembodiments a protocol can be dependent on a fluidic device identifier.In some embodiments the external device stores more than one protocolfor each fluidic device. In other embodiments patient information on theexternal device includes more than one protocol. In preferredembodiments the external server stores mathematical algorithms toprocess a photon count sent from a communication assembly and in someembodiments to calculate the analyte concentration in a bodily fluidsample.

In some embodiment the external device can include one or more serversas are known in the art and commercially available. Such servers canprovide load balancing, task management, and backup capacity in theevent of failure of one or more of the servers or other components ofthe external device, to improve the availability of the server. A servercan also be implemented on a distributed network of storage andprocessor units, as known in the art, wherein the data processingaccording to the present invention reside on workstations such ascomputers, thereby eliminating the need for a server.

A server can includes a database and system processes. A database canreside within the server, or it can reside on another server system thatis accessible to the server. As the information in a database maycontains sensitive information, a security system can be implementedthat prevents unauthorized users from gaining access to the database.

One advantage of the present invention is that information can betransmitted from the external device back to not only the readerassembly, but to other parties or other external devices, for examplewithout limitation, a PDA or cell phone. Such communication can beaccomplished via a wireless network as disclosed herein. In someembodiments a calculated analyte concentration or other patientinformation can be sent to, for example but not limited to, medicalpersonal or the patient.

Method of Use

The subject apparatus and systems provide an effective means for highthroughput and real-time detection of analytes present in a bodily fluidfrom a subject. The detection methods may be used in a wide variety ofcircumstances including identification and quantification of analytesthat are associated with specific biological processes, physiologicalconditions, disorders or stages of disorders. As such, the subjectapparatus and systems have a broad spectrum of utility in, e.g. drugscreening, disease diagnosis, phylogenetic classification, parental andforensic identification. The subject apparatus and systems are alsoparticularly useful for advancing preclinical and clinical stage ofdevelopment of therapeutics, improving patient compliance, monitoringADRs associated with a prescribed drug, and developing individualizedmedicine.

Accordingly, in one embodiment, the present invention provides a methodof detecting an analyte in a bodily fluid from a subject comprisesproviding a fluidic device comprising at least one sample collectionunit, an immunoassay assembly containing immunoassay reagents, aplurality of channels in fluid communication with said sample collectionunit and/or said immunoassay assembly; actuating said fluidic device anddirecting said immunoassay reagents within said fluidic device; allowinga sample of bodily fluid to react with said immunoassay reagentscontained within said assay immunoassay assembly to yield a detectablesignal indicative of the presence of said analyte in said bodily fluid;and detecting said detectable signal generated from said analyteinitially collected in said sample of bodily fluid. Preferably, a sampleof bodily fluid of less than about 1 ml, preferably less than about 500ul is used for one or more of these applications.

As used herein, the term “subject” or “patient” is used interchangeablyherein, which refers to a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, murines,simians, humans, farm animals, sport animals, and pets.

In some embodiments a sample of bodily fluid can first be provided tothe fluidic device by any of the methods described herein. The fluidicdevice can then be inserted into the reader assembly. An identificationdetector housed within the reader assembly can detect an identifier ofthe fludic device and communicate the identifier to a communicationassembly, which is preferably housed within the reader assembly. Thecommunication assembly then transmits the identifier to an externaldevice which transmits a protocol to run on the fluidic device based onthe identifier to the communication assembly. A controller preferablyhoused within the reader assembly controls actuating elements includingat least one pump and one valve which interact with the fluidic deviceto control and direct fluid movement within the device. In someembodiments the first step of the assay is a wash cycle where all thesurfaces within the fluidic device are wetted using a wash buffer. Thefluidic device is then calibrated using a calibration assembly byrunning the same reagents as will be used in the assay through thecalibration reaction sites, and then a luminescence signal from thereactions sites is detected by the detection means, and the signal isused in calibrating the fluidic device. The sample containing theanalyte is introduced into the fluidic channel. The sample may bediluted and further separated into plasma or other desired component ata filter. The separated sample now flows through the reaction sites andanalytes present therein will bind to reactants bound thereon. Theplasma of sample fluid is then flushed out of the reaction wells into awaste chamber. Depending on the assay being run, appropriate reagentsare directed through the reaction sites to carry out the assay. All thewash buffers and other reagents used in the various steps, including thecalibration step, are collected in wash tanks. The signal produced inthe reaction sites is then detected by any of the methods describedherein.

A variety of assays may be performed on a fluidic device according tothe present invention to detect an analyte of interest in a sample. Awide diversity of labels are available in the art that can be employedfor conducting the subject assays. In some embodiments labels aredetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. For example, useful nucleic acid labels include 32P,35S, fluorescent dyes, electron-dense reagents, enzymes, biotin,dioxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available. A wide variety of labels suitable for labelingbiological components are known and are reported extensively in both thescientific and patent literature, and are generally applicable to thepresent invention for the labeling of biological components. Suitablelabels include radionucleotides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties,bioluminescent labels, calorimetric labels, or magnetic particles.Labeling agents optionally include, for example, monoclonal antibodies,polyclonal antibodies, proteins, or other polymers such as affinitymatrices, carbohydrates or lipids. Detection proceeds by any of avariety of known methods, including spectrophotometric or opticaltracking of radioactive or fluorescent markers, or other methods whichtrack a molecule based upon size, charge or affinity. A detectablemoiety can be of any material having a detectable physical or chemicalproperty. Such detectable labels have been well-developed in the fieldof gel electrophoresis, column chromatography, solid substrates,spectroscopic techniques, and the like, and in general, labels useful insuch methods can be applied to the present invention. Thus, a labelincludes without limitation any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical thermal,or chemical means.

In some embodiments the label is coupled directly or indirectly to amolecule to be detected such as a product, substrate, or enzyme,according to methods well known in the art. As indicated above, a widevariety of labels are used, with the choice of label depending on thesensitivity required, ease of conjugation of the compound, stabilityrequirements, available instrumentation, and disposal provisions. Nonradioactive labels are often attached by indirect means. Generally, aligand molecule is covalently bound to a polymer. The ligand then bindsto an anti-ligand molecule which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. A number ofligands and anti-ligands can be used. Where a ligand has a naturalanti-ligand, for example, biotin, thyroxine, and cortisol, it can beused in conjunction with labeled, anti-ligands. Alternatively, anyhaptenic or antigenic compound can be used in combination with anantibody.

In some embodiments the label can also be conjugated directly to signalgenerating compounds, for example, by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,and umbelliferone. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, such as luminol.

Methods of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence by, for example,microscopy, visual inspection, via photographic film, by the use ofelectronic detectors such as digital cameras, charge coupled devices(CCDs) or photomultipliers and phototubes, or other detection device.Similarly, enzymatic labels are detected by providing appropriatesubstrates for the enzyme and detecting the resulting reaction product.Finally, simple colorimetric labels are often detected simply byobserving the color associated with the label. For example, conjugatedgold often appears pink, while various conjugated beads appear the colorof the bead.

In some embodiments the detectable signal may be provided byluminescence sources. “Luminescence” is the term commonly used to referto the emission of light from a substance for any reason other than arise in its temperature. In general, atoms or molecules emit photons ofelectromagnetic energy (e.g., light) when then move from an “excitedstate” to a lower energy state (usually the ground state); this processis often referred to as “radioactive decay”. There are many causes ofexcitation. If exciting cause is a photon, the luminescence process isreferred to as “photoluminescence”. If the exciting cause is anelectron, the luminescence process is referred to as“electroluminescence”. More specifically, electroluminescence resultsfrom the direct injection and removal of electrons to form anelectron-hole pair, and subsequent recombination of the electron-holepair to emit a photon. Luminescence which results from a chemicalreaction is usually referred to as “chemiluminescence”. Luminescenceproduced by a living organism is usually referred to as“bioluminescence”. If photoluminescence is the result of a spin-allowedtransition (e.g., a single-singlet transition, triplet-triplettransition), the photoluminescence process is usually referred to as“fluorescence”. Typically, fluorescence emissions do not persist afterthe exciting cause is removed as a result of short-lived excited stateswhich may rapidly relax through such spin-allowed transitions. Ifphotoluminescence is the result of a spin-forbidden transition (e.g., atriplet-singlet transition), the photoluminescence process is usuallyreferred to as “phosphorescence”. Typically, phosphorescence emissionspersist long after the exciting cause is removed as a result oflong-lived excited states which may relax only through suchspin-forbidden transitions. A “luminescent label” may have any one ofthe above-described properties.

Suitable chemiluminescent sources include a compound which becomeselectronically excited by a chemical reaction and may then emit lightwhich serves as the detectable signal or donates energy to a fluorescentacceptor. A diverse number of families of compounds have been found toprovide chemiluminescence under a variety or conditions. One family ofcompounds is 2,3-dihydro-1,4-phthalazinedione. A frequently usedcompound is luminol, which is a 5-amino compound. Other members of thefamily include the 5-amino-6,7,8-trimethoxy- and thedimethylamino[ca]benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, for example, p-nitrophenyl and a peroxide such ashydrogen peroxide, under basic conditions. Other useful chemiluminescentcompounds that are also known include —N-alkyl acridinum esters anddioxetanes. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins to provide bioluminescence.

In some embodiments immunoassays are run on the fluidic device. Whilecompetitive binding assays, which are well known in the art, may be runin some embodiments, in preferred embodiments a two-step method is usedwhich eliminates the need to mix a conjugate and a sample beforeexposing the mixture to an antibody, which may be desirable when verysmall volumes of sample and conjugate are used, as in the fluidic deviceof the present invention. A two-step assay has additional advantagesover the competitive binding assays when use with a fluidic device asdescribed herein. It combines the ease of use and high sensitivity of asandwich (competitive binding) immunoassay with the ability to assaysmall molecules.

In an exemplary two-step assay shown in FIG. 10, the sample containinganalyte (“Ag”) first flows over a reaction site containing antibodies(“Ab”). The antibodies bind the analyte present in the sample. After thesample passes over the surface, a solution with analyte conjugated to amarker (“labeled Ag”) at a high concentration is passed over thesurface. The conjugate saturates any of the antibodies that have not yetbound the analyte. Before equilibrium is reached and any displacement ofpre-bound unlabelled analyte occurs, the high-concentration conjugatesolution is washed off. The amount of conjugate bound to the surface isthen measured by the appropriate technique, and the detected conjugateis inversely proportional to the amount of analyte present in thesample.

An exemplary measuring technique for a two-step assay is achemiluminescence enzyme immunoassay as shown in FIG. 11. As is known inthe field, the marker can be a commercially available marker such asdioxitane-phosphate, which is not luminescent but becomes luminescentafter hydrolysis by, for example, alkaline phosphatase. An enzyme suchas alkaline phosphatase is also passed over the substrate to cause themarker to luminesce. In some embodiments the substrate solution issupplemented with enhancing agents such as, without limitation,fluorescein in mixed micelles, soluble polymers, or PVC which create amuch brighter signal than the luminophore alone. Moreover, an alkalinephosphatase conjugate with a higher turnover number than that used inthe commercial assay is employed. This allows signal generation toproceed much more rapidly and a higher overall signal is achieved. Theincreased sensitivity of the two-step chemiluminescent enzymeimmunoassay (TOSCA) is illustrated in FIG. 12. FIG. 12 shows that foranalytes in the picomolar concentration, TOSCA is able to provide a morerobust signal (higher sensitivity) than a competitive binding assay. Useof a two-step binding assay thus contributes to higher sensitivitycapabilities of the present invention.

Additionally, TOSCA is less sensitive to matrix effects than othermethodologies. This allows one to work with samples that have not beenextensively pre-processed using standard laboratory techniques such as,for example, solid phase extraction and chromatography. The ability ofTOSCA to assay less than ideal samples and maintain desired sensitivityis illustrated in FIG. 13. Compared to competitive binding assay, forall sample preparations (and dilutions), TOSCA has better sensitivitythan competitive binding. This is also illustrated in FIG. 17 where thesensitivity enhancement achieved using TOSCA is compared with thetwo-step assay.

The term “analytes” according to the present invention includes withoutlimitation drugs, prodrugs, pharmaceutical agents, drug metabolites,biomarkers such as expressed proteins and cell markers, antibodies,serum proteins, cholesterol, polysaccharides, nucleic acids, biologicalanalytes, biomarker, gene, protein, or hormone, or any combinationthereof. At a molecular level, the analytes can be polypeptideglycoprotein, polysaccharide, lipid, nucleic acid, and a combinationthereof.

Of particular interest are biomarkers are associated with a particulardisease or with a specific disease stage. Such analytes include but arenot limited to those associated with autoimmune diseases, obesity,hypertension, diabetes, neuronal and/or muscular degenerative diseases,cardiac diseases, endocrine disorders, any combinations thereof.

Of also interest are biomarkers that are present in varying abundance inone or more of the body tissues including heart, liver, prostate, lung,kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, andselected tissues that are affected by various disease, such as differenttypes of cancer (malignant or non-metastatic), autoimmune diseases,inflammatory or degenerative diseases.

Also of interest are analytes that are indicative of a microorganism.Exemplary microorganisms include but are not limited to bacterium,virus, fungus and protozoa. Analytes that can be detected by the subjectmethod also include blood-born pathogens selected from a non-limitinggroup that consists of Staphylococcus epidermidis, Escherichia coli,methicillin-resistant Staphylococcus aureus (MSRA), Staphylococcusaureus, Staphylococcus hominis, Enterococcus faecalis, Pseudomonasaeruginosa, Staphylococcus capitis, Staphylococcus warneri, Klebsiellapneumoniae, Haemophilus influnzae, Staphylococcus simulans,Streptococcus pneumoniae and Candida albicans.

Analytes that can be detected by the subject method also encompass avariety of sexually transmitted diseases selected from the following:gonorrhea (Neisseria gorrhoeae), syphilis (Treponena pallidum), clamydia(Clamyda tracomitis), nongonococcal urethritis (Ureaplasm urealyticum),yeast infection (Candida albicans), chancroid (Haemophilus ducreyi),trichomoniasis (Trichomonas vaginalis), genital herpes (HSV type I &II), HIV I, HIV II and hepatitis A, B, C, G, as well as hepatitis causedby TTV.

Additional analytes that can be detected by the subject methodsencompass a diversity of respiratory pathogens including but not limitedto Pseudomonas aeruginosa, methicillin-resistant Staphlococccus aureus(MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphlococcusaureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae,Escherichia coli, Enterococcus faecalis, Serratia marcescens,Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans,Moraxiella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii,Enterococcus faecium, Klebsella oxytoca, Pseudomonas fluorscens,Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii,Klebsella pneumoniae Legionella pneumophila, Mycoplasma pneumoniae, andMycobacterium tuberculosis.

Listed below are additional exemplary markers according to the presentinvention: Theophylline, CRP, CKMB, PSA, Myoglobin, CA125, Progesterone,TxB2,6-keto-PGF-1-alpha, and Theophylline, Estradiol, Lutenizinghormone, High sensitivity CRP, Triglycerides, Tryptase, Low densitylipoprotein Cholesterol, High density lipoprotein Cholesterol,Cholesterol, IGFR.

Exemplary liver markers include without limitation LDH, (LD5), (ALT),Arginase 1 (liver type), Alpha-fetoprotein (AFP), Alkaline phosphatase,Alanine aminotransferase, Lactate dehydrogenase, and Bilirubin.

Exemplary kidney markers include without limitation TNFa Receptor,Cystatin C, Lipocalin-type urinary prostaglandin D, synthatase (LPGDS),Hepatocyte growth factor receptor, Polycystin 2, Polycystin 1,Fibrocystin, Uromodulin, Alanine, aminopeptidase,N-acetyl-B-D-glucosaminidase, Albumin, and Retinol-binding protein(RBP).

Exemplary heart markes include without limitation Troponin I (TnI),Troponin T (TnT), CK, CKMB, Myoglobin, Fatty acid binding protein(FABP), CRP, D-dimer, S-100 protein, BNP, NT-proBNP, PAPP-A,Myeloperoxidase (MPO), Glycogen phosphorylase isoenzyme BB (GPBB),Thrombin Activatable Fibrinolysis Inhibitor (TAFI), Fibrinogen, Ischemiamodified albumin (IMA), Cardiotrophin-1, and MLC-I (Myosin LightChain-I).

Exemplary pancrease markers include without limitation Amylase,Pancreatitis-Assocoated protein (PAP-1), and Regeneratein proteins(REG).

Exemplary muscle tissue markers include without limitation Myostatin.

Exemplary blood markers include without limitation Erythopoeitin (EPO).

Exemplary bone markers include without limitation, Cross-linkedN-telopeptides of bone type I collagen (NTx)

Carboxyterminal cross-linking telopeptide of bone collagen,Lysyl-pyridinoline (deoxypyridinoline), Pyridinoline, Tartrate-resistantacid phosphatase, Procollagen type I C propeptide, Procollagen type 1Npropeptide, Osteocalcin (bone gla-protein), Alkaline phosphatase,Cathepsin K, COMP (Cartillage Oligimeric Matrix Protein), OsteocrinOsteoprotegerin (OPG), RANKL, sRANK, TRAP 5 (TRACP 5), OsteoblastSpecific Factor 1 (OSF-1, Pleiotrophin), Soluble cell adhesionmolecules, sTfR, sCD4, sCD8, sCD44, and Osteoblast Specific Factor 2(OSF-2, Periostin).

In some embodiments markers according to the present invention aredisease specific. Exemplary cancer markers include without limitationPSA (total prostate specific antigen), Creatinine, Prostatic acidphosphatase, PSA complexes, Prostrate-specific gene-1, CA 12-5,Carcinoembryonic Antigen (CEA), Alpha feto protein (AFP), hCG (Humanchorionic gonadotropin), Inhibin, CAA Ovarian C1824, CA 27.29, CA 15-3,CAA Breast C1924, Her-2, Pancreatic, CA 19-9, Carcinoembryonic Antigen,CAA pancreatic, Neuron-specific enolase, Angiostatin

DcR3 (Soluble decoy receptor 3), Endostatin, Ep-CAM (MK-1), FreeImmunoglobulin Light Chain Kappa, Free Immunoglobulin Light ChainLambda, Herstatin, Chromogranin A, Adrenomedullin, Integrin, Epidermalgrowth factor receptor, Epidermal growth factor receptor-Tyrosinekinase, Pro-adrenomedullin N-terminal 20 peptide, Vascular endothelialgrowth factor, Vascular endothelial growth factor receptor, Stem cellfactor receptor, c-kit/KDR, KDR, and Midkine.

Exemplary infectious disease markers include without limitation Viremia,Bacteremia, Sepsis, PMN Elastase, PMN elastase/α1-PI complex, SurfactantProtein D (SP-D), HBVc antigen, HBVs antigen, Anti-HBVc, Anti-HIV,T-supressor cell antigen, T-cell antigen ratio, T-helper cell antigen,Anti-HCV, Pyrogens, p24 antigen, Muramyl-dipeptide.

Exemplary diabetes markers include without limitation C-Peptide,Hemoglobin A1c, Glycated albumin, Advanced glycosylation end products(AGEs), 1,5-anhydroglucitol, Gastric Inhibitory Polypeptide, Glucose,Hemoglobin, ANGPTL3 and 4.

Exemplary inflammation markers include without limitation Rheumatoidfactor (RF), Antinuclear Antibody (ANA), C-reactive protein (CRP), ClaraCell Protein (Uteroglobin).

Exemplary allergy markers include without limitation Total IgE andSpecific IgE.

Exemplary autism markers include without limitation Ceruloplasmin,Metalothioneine, Zinc, Copper, B6, B12, Glutathione, Alkalinephosphatase, and Activation of apo-alkaline phosphatase.

Exemplary coagulation disorders markers include without limitationb-Thromboglobulin, Platelet factor 4, Von Willebrand factor.

In some embodiments a marker may be therapy specific. COX inhibitorsinclude without limitation TxB2 (Cox-1), 6-keto-PGF-1-alpha (Cox 2),11-Dehydro-TxB-1a (Cox-1).

Other markers of the present include without limitation Leptin, Leptinreceptor, and Procalcitonin, Brain S100 protein, Substance P,8-Iso-PGF-2a.

Exemplary geriatric markers include without limitation, Neuron-specificenolase, GFAP, and S100B.

Exemplary markers of nutritional status include without limitationPrealbumin, Albumin, Retinol-binding protein (RBP), Transferrin,Acylation-Stimulating Protein (ASP), Adiponectin, Agouti-Related Protein(AgRP), Angiopoietin-like Protein 4 (ANGPTL4, FIAF), C-peptide, AFABP(Adipocyte Fatty Acid Binding Protein, FABP4)

Acylation-Stimulating Protein (ASP), EFABP (Epidermal Fatty Acid BindingProtein, FABP5), Glicentin, Glucagon, Glucagon-Like Peptide-1,Glucagon-Like Peptide-2, Ghrelin, Insulin, Leptin, Leptin Receptor, PYY,RELMs, Resistin, amd sTfR (soluble Transferrin Receptor).

Exemplary markers of Lipid metabolism include without limitationApo-lipoproteins (several), Apo-A1, Apo-B, Apo-C-CII, Apo-D, Apo-E.

Exemplary coagulation status markers include without limitation FactorI: Fibrinogen, Factor II: Prothrombin, Factor III: Tissue factor, FactorIV: Calcium, Factor V: Proaccelerin, Factor VI, Factor VII:Proconvertin, Factor VIII: Anti-hemolytic factor, Factor IX: Christmasfactor, Factor X: Stuart-Prower factor, Factor XI: Plasma thromboplastinantecedent, Factor XII: Hageman factor, Factor XIII: Fibrin-stabilizingfactor, Prekallikrein, High-molecular-weight kininogen, Protein C,Protein S, D-dimer, Tissue plasminogen activator, Plasminogen,a2-Antiplasmin, Plasminogen activator inhibitor 1 (PAI1).

Exemplary monoclonal antibodies include those for EGFR, ErbB2, andIGF1R.

Exemplary tyrosine kinase inhibitors include without limitation Ab1,Kit, PDGFR, Src, ErbB2, ErbB 4, EGFR, EphB, VEGFR1-4, PDGFRb, FLt3,FGFR, PKC, Met, Tie2, RAF, and TrkA.

Exemplary Serine/Threoline Kinas Inhibitors include without limitationAKT, Aurora A/B/B, CDK, CDK (pan), CDK1-2, VEGFR2, PDGFRb, CDK4/6,MEK1-2, mTOR, and PKC-beta.

GPCR targets include without limitation Histamine Receptors, SerotoninReceptors, Angiotensin Receptors, Adrenoreceptors, MuscarinicAcetylcholine Receptors, GnRH Receptors, Dopamine Receptors,Prostaglandin Receptors, and ADP Receptors.

In a separate embodiment, the present invention provides a method ofmonitoring more than one pharmacological parameter useful for assessingefficacy and/or toxicity of a therapeutic agent. The method comprisessubjecting a sample of bodily fluid from a subject administered with thetherapeutic agent to a fluidic device for monitoring said more than onepharmacological parameter, said fluidic device comprising at least onesample collection unit, and an assay assembly comprising reactionreagents; actuating said fluidic device and directing said immunoassayreagents within said fluidic device; allowing said sample of bodilyfluid to react with immunoassay reagents to yield detectable signalsindicative of the values of the more than one pharmacological parameterfrom said sample; and detecting said detectable signal generated fromsaid sample of bodily fluid. Where desired, the method further involvesrepeating the steps at a time interval prompted by a wireless signalcommunicated to the subject.

For the purposes of this invention, a “therapeutic agent” is intended toinclude any substances that have therapeutic utility and/or potential.Such substances include but are not limited to biological or chemicalcompounds such as a simple or complex organic or inorganic molecules,peptides, proteins (e.g. antibodies) or a polynucleotides (e.g.anti-sense). A vast array of compounds can be synthesized, for examplepolymers, such as polypeptides and polynucleotides, and syntheticorganic compounds based on various core structures, and these are alsoincluded in the term “therapeutic agent”. In addition, various naturalsources can provide compounds for screening, such as plant or animalextracts, and the like. It should be understood, although not alwaysexplicitly stated that the agent is used alone or in combination withanother agent, having the same or different biological activity as theagents identified by the inventive screen. The agents and methods alsoare intended to be combined with other therapies.

Pharmacodynamic (PD) parameters according to the present inventioninclude without limitation physical parameters such as temperature,heart rate/pulse, blood pressure, and respiratory rate, and biomarkerssuch as proteins, cells, and cell markers. Biomarkers could beindicative of disease or could be a result of the action of a drug.Pharmacokinetic (PK) parameters according to the present inventioninclude without limitation drug and drug metabolite concentration.Identifying and quantifying the PK parameters in real time from a samplevolume is extremely desirable for proper safety and efficacy of drugs.If the drug and metabolite concentrations are outside a desired rangeand/or unexpected metabolites are generated due to an unexpectedreaction to the drug, immediate action may be necessary to ensure thesafety of the patient. Similarly, if any of the pharmacodynamic (PD)parameters fall outside the desired range during a treatment regime,immediate action may have to be taken as well.

In preferred embodiments physical parameter data is stored in orcompared to store profiles of physical parameter data in abioinformatics system which may be on an external device incorporatingpharmacogenomic and pharmacokinetic data into its models for thedetermination of toxicity and dosing. Not only does this generate datafor clinical trials years prior to current processes but also enablesthe elimination of current disparities between apparent efficacy andactual toxicity of drugs through real-time continuous monitoring. Duringthe go/no go decision process in clinical studies, large scalecomparative population studies can be conducted with the data stored onthe database. This compilation of data and real-time monitoring allowsmore patients to enter clinical trials in a safe fashion earlier thancurrently allowed. In another embodiment biomarkers discovered in humantissue studies can be targeted by the device for improved accuracy indetermining drug pathways and efficacy in cancer studies.

In another embodiment, the present invention provides a method ofdetecting at least two distinct analytes of different concentrations ina bodily fluid from a subject comprises providing a fluidic devicecomprising a sample collection unit, an assay assembly, and a pluralityof channels in fluid communication with said sample collection unitand/or said assay assembly; allowing a sample of bodily fluid to reactwith a plurality of reactants contained in said assay assembly to yieldsignals indicative of the concentrations of said at least two analytes;and detecting said signals that are indicative of the presence orabsence of the at least two distinct analytes, wherein said signals aredetectable over a range of 3 orders of magnitude.

Currently, a need exists for the detecting more than one analyte wherethe analytes are present in widely varying concentration range, forexample, one analyte is in the pg/ml concentration and another is in theng/ml concentration. TOSCA described herein has the ability tosimultaneously assay analytes that are present in the same sample in awide concentration range. FIG. 18 shows one embodiment where twoanalytes, prostacyclin metabolite and thromboxane metabolite, have beenidentified and quantified and their concentrations are different by morethan 3 orders of magnitude. Another advantage for being able to detectconcentrations of different analytes present in a wide concentrationrange is the ability to relate the ratios of the concentration of theseanalytes to safety and efficacy of multiple drugs administered to apatient. For example, unexpected drug-drug interactions can be a commoncause of adverse drug reactions. A real-time, concurrent measurementtechnique for measuring different analytes would help avoid thepotentially disastrous consequence of adverse drug-drug interactions.

Being able to monitoring the rate of change of an analyte concentrationor PD or PK over a period of time in a single subject, or performingtrend analysis on the concentration, PD, or PK, whether they areconcentrations of drugs or their metabolites, can help preventpotentially dangerous situations. For example, if glucose were theanalyte of interest, the concentration of glucose in a sample at a giventime as well as the rate of change of the glucose concentration over agiven period of time could be highly useful in predicting and avoiding,for example, hypoglycemic events. Such trend analysis has widespreadbeneficial implications in drug dosing regimen. When multiple drugs andtheir metabolites are concerned, the ability to spot a trend and takeproactive measures is often desirable.

Accordingly, the present invention provides a method of performing atrend analysis on the concentration of an analyte in a subject. Themethod comprise a) providing a fluidic device comprising at least onesample collection unit, an immunoassay assembly containing immunoassayreagents, a plurality of channels in fluid communication with saidsample collection unit and/or said immunoassay assembly; b) actuatingsaid fluidic device and directing said immunoassay reagents within saidfluidic device; c) allowing a sample of bodily fluid of less than about500 ul to react with said immunoassay reagents contained within saidassay immunoassay assembly to yield a detectable signal indicative ofthe presence of said analyte in said sample; d) detecting saiddetectable signal generated from said analyte collected in said sampleof bodily fluid; and e) repeating steps a) through d) for a singlepatient over a period of time to detect concentrations of said anayte,thereby performing said trend analysis.

In some embodiments, a method of detecting an analyte in a bodily fluidfrom a subject using an assay transmitted from an external device isprovided. The method comprises providing a fluidic device comprising atleast one sample collection unit and an immunoassay assembly containingimmunoassay reagents; detecting said fluidic device and wirelesslytransmitting an immunoassay protocol to said device; allowing a sampleof bodily fluid to react with immunoassay reagents to yield a detectablesignal indicative of the presence of said analyte using said transmittedimmunoassay protocol; and detecting said detectable signal.

Communication between a reader assembly and an external storage deviceallows for a reader assembly of the present invention to download afluidic device-specific protocol to run on the fluidic device based onthe identity of the fluidic device. This allows a reader assembly to beused interchangeably with any appropriate fluidic device describedherein. In addition, the external device can store a plurality ofprotocols associated with a given fluidic device, and depending on, forexample, a subject's treatment regime or plan, different protocols canbe communicated from the external device to the reader assembly to berun on the fluidic device to detect a variety of analytes. The externaldevice can also store a plurality of protocols associated not only witha fluidic device, but also with a particular subject or subjects, suchthat a protocol can be associated with a subject as well as with afluidic device.

In some embodiments, the present invention provides a business method ofassisting a clinician in providing an individualized medical treatmentcomprises collecting at least one pharmacological parameter from anindividual receiving a medication, said collecting step is effected bysubjecting a sample of bodily fluid to reactants contained in a fluidicdevice, which is provided to said individual to yield a detectablesignal indicative of said at least one pharmacological parameter; andcross referencing with the aid of a computer medical records of saidindividual with the at least one pharmacological parameter of saidindividual, thereby assisting said clinician in providing individualizedmedical treatment.

The present invention allows for automatic quantification of apharmacological parameter of a patient as well as automatic comparisonof the parameter with, for example, the patient's medical records whichmay include a history of the monitored parameter, or medical records ofanother group of subjects. Coupling real-time analyte monitoring with anexternal device which can store data as well as perform any type of dataprocessing or algorithm, for example, provides a device that can assistwith typical patient care which can include, for example, comparingcurrent patient data with past patient data. The present inventiontherefore creates a business method which effectively performs at leastpart of the monitoring of a patient that is currently performed bymedical personnel.

In some embodiments, the present invention provides a business method ofmonitoring a clinical trial of a pharmaceutical agent comprisescollecting at least one pharmacological parameter from a subject in saidclinical trial at a plurality of time intervals, said collecting step iseffected at each time interval by subjecting a sample of bodily fluidfrom said subject to reactants contained in a fluidic device, whereinsaid fluidic device is provided to said subject to yield detectablesignals indicative of the values of said at least one pharmacologicalparameter at a plurality of time intervals; comparing the detectedvalues to a threshold value predetermined for said pharmacologicalparameter; notifying a clinician and/or a sponsor involved in saidclinical trial when a statistically significant discrepancy existsbetween the detected values and the threshold value.

FIG. 19 shows an exemplary flow chart of a business method of monitoringa clinical trial of a pharmaceutical agent. As disclosed herein, afluidic device gathers PK and/or PD parameters related to a patient ofinterest. The data is securely transmitted over, for example, a cellularnetwork or the internet, and interpretations of the data are derivedthrough computations in a series of biostatistical algorithms on theexternal device which correlate pharmacodynamic, pharmacokinetic, andpharmacogenetic profiles. Additionally, the data can be compared withinformation stored in databases. The stored information could be thepatient's own PK and PD data over a previous treatment regiment, datarelated to placebo, pharmacogenomic data that are of relevance to theparticular patient, or data related to a group of subjects. If theanalysis done in Step 2 suggests that there are no significantdifference between the patient's data and the stored data, as determinedby using appropriate algorithms, then “No Action” is taken. However, ifthere is a significant difference, then Step 4 determines the size ofthe difference. If the difference is large, immediate action is taken.An exemplary type of immediate action could be to provide an emergencyalert to the patient's healthcare provider. Another kind of immediateaction could be to send instructions to the fluidic device to alter thedosing of the pharmaceutical agent. If in Step 4 the difference issmall, then the algorithm could determine whether to continue monitoringthe parameters and/or alter a dosage of the pharmaceutical agent. Thismethod provides for automatic notification to at least medical personnelor a subject of a possible need to take additional medical action.

Where a statistically significant discrepancy exists between thedetected values and the threshold value, a further action may be takenby a medical practitioner. Such action may involve a medical action suchas adjusting dosage of the therapeutic agent; it may also involve abusiness decision such as continuing, modifying, or terminating theclinical trial.

One of the significant advantages of the envisioned network isillustrated in FIG. 20. As all the information is securely channeledthrough the internet, this allows the simultaneous sharing of theinformation with various interested parties, while satisfying theappropriate clinical, regulatory and business needs. For example, theflowchart shows how the patient's clinical needs are met. The ability ofthe company that is sponsoring a drug study, for example a clinicaltrial or a post-market Phase IV surveillance, to monitor in real-timethe safety and efficacy of the performance of the drug providesextremely valuable regulatory and business information. Similarly, theability of a payor to monitor the efficacy, and perhapscost-effectiveness, of a treatment is greatly enhanced by their abilityto obtain data in real-time.

In some embodiments, the present invention provides a method oftransmitting a pharmacological parameter of a patient via a handhelddevice comprises providing a fluidic device comprising at least onesample collection unit and an assay assembly; allowing a sample ofbodily fluid to react with reactants contained within said assayassembly to yield a detectable signal indicative of the presence of saidanalyte; detecting said detectable signal; transmitting said signal toan external device; processing said signal in said external device; andtransmitting said processed signal via a handheld device.

One advantage of the current invention is that assay results can besubstantially immediately communicated to any third party that maybenefit from obtaining the results. For example, once the analyteconcentration is determined at the external device, it can betransmitted to a patient or medical personnel who may need to takefurther action. The communication step to a third party can be performedwirelessly as described herein, and by transmitting the data to a thirdparty's hand held device, the third party can be notified of the assayresults virtually anytime and anywhere. Thus, in a time-sensitivescenario, a patient may be contacted immediately anywhere if urgentmedical action may be required.

In some embodiments a method of automatically selecting a protocol to berun on a fluidic device comprises providing a fluidic device comprisingan identifier detector and an identifier; detecting said identifier withsaid identifier detector; transferring said identifier to an externaldevice; and selecting a protocol to be run on said fluidic device from aplurality of protocols on said external device associated with saididentifier.

By detecting each fluidic device based on an identifier associated withthe fluidic device after it is inserted in the reader assembly, thesystem of the present invention allows for fluidic device-specificprotocols to be downloaded from an external device and run on thefluidic device. In some embodiments the external device can store aplurality of protocols associated with the fluidic device or associatedwith a particular patient or group of patients. For example, when theidentifier is transmitted to the external device, software on theexternal device can obtain the identifier. Once obtained, software onthe external device, such as a database, can use the identifier toidentify protocols stored in the database associated with theidentifier. If only one protocol is associated with the identifier, forexample, the database can select the protocol and software on theexternal device can then transmit the protocol to the communicationassembly on the reader assembly. The ability to use protocolsspecifically associated with a fluidic device allows for any appropriatefluidic device to be used with a single reader assembly, and thusvirtually any analyte of interest can be detected with a single readerassembly.

In some embodiments multiple protocols may be associated with a singleidentifier. For example, if it is beneficial to detect from the samepatient an analyte once a week, and another analyte twice a week,protocols on the external device associated with the identifier can alsoeach be associated with a different day of the week, so that when theidentifier is detected, the software on the external device can select aspecific protocol that is associated with the day of the week.

In some embodiments a patient may be provided with a plurality offluidic devices to use to detect a variety of analytes. A subject may,for example, use different fluidic devices on different days of theweek. In some embodiments the software on the external deviceassociating the identifier with a protocol may include a process tocompare the current day with the day the fluidic device is to be usedbased on a clinical trial for example. If for example, the two days ofthe week are not identical, the external device can wirelessly sendnotification to the subject using any of the methods described herein orknown in the art to notify them that an incorrect fluidic device is inthe reader assembly and also of the correct fluidic device to use thatday. This example is only illustrative and can easily be extended to,for example, notifying a subject that a fluidic device is not being usedat the correct time of day.

In some embodiments, the present invention provides a method ofmanufacturing a fluidic device for detecting an analyte in a biologicalfluid of a subject comprises providing a plurality of layers of amaterial. The method comprises providing a plurality of layers of afluidic device, and ultrasonically welding said layers together suchthat a fluidic network exists between a sample collection unit, at leastone reactant chamber, at least one reaction site, and at least one wastechamber. Where desired, the fluidic device manufactured by this methodcomprise in at least one of said layers a sample collection unit, atleast one of said layers comprises a filtration site, and at least oneof said layers comprises a reactant chamber, and at least one of saidlayers comprises a fluidic channel, and at least one of said layerscomprises a reaction site, and at least one of said layers comprises awaste chamber.

In preferred embodiments the different layers of the fluidic device areultrasonically welded together according to methods known in the art.The layers may also be coupled together using other methods, includingwithout limitation stamping, thermal bonding, adhesives or, in the caseof certain substrates, e.g., glass, or semi-rigid and non-rigidpolymeric substrates, a natural adhesion between the two components.

In some embodiments, the present invention provides a method ofobtaining pharmacological data useful for assessing efficacy and/ortoxicity of a pharmaceutical agent from a test animal. The methodinvolves the steps of a) providing a fluidic device comprising at leastone sample collection unit, an assay assembly; and a plurality ofchannels in fluid communication with said sample collection unit and/orsaid assay assembly; b) allowing a sample of biological fluid of lessthan about 50 ul to react with reactants contained within said assayassembly to yield a detectable signal generated from an analyteinitially collected in said sample that is indicative of apharmacological parameter; and c) detecting said detectable signal; andd) repeating the reaction and detection steps with a second sample ofbiological fluid from the same test animal. In a related embodiment, thepresent invention provides a method comprising a) providing a fluidicdevice comprising at least one sample collection unit, an assayassembly; and a plurality of channels in fluid communication with saidsample collection unit and/or said assay assembly; b) allowing a sampleof biological fluid to react with reactants contained within said assayassembly to yield a detectable signal generated from an analyteinitially collected in said sample that is indicative of apharmacological parameter; and c) detecting said detectable signal; andd) repeating the reaction and detection steps with a second sample ofbiological fluid from the same test animal, wherein the animal is notsubjected to anesthesia.

When using laboratory animals in preclinical testing of a pharmaceuticalagent, it is often necessary to kill the test subject to extract enoughblood to perform an assay to detect an analyte of interest. This hasboth financial and ethical implications, and as such it may beadvantageous to be able to draw an amount of blood from a test animalsuch that the animal does not need to be killed. In addition, this canalso allow the same test animal to be tested with multiplepharmaceutical agents at different times, thus allowing for a moreeffective preclinical trial. On average, the total blood volume in amouse, for example, is 6-8 mL of blood per 100 gram of body weight. Abenefit of the current invention is that only a very small volume ofblood is required to perform preclinical trials on mice or other smalllaboratory animals. In some embodiment between about 1 microliter andabout 50 microliters are drawn. In preferred embodiment between about 1microliter and 10 microliters are drawn. In preferred embodiments about5 microliters of blood are drawn.

A further advantage of keeping the test animal alive is evident in apreclinical time course study. When multiple mice, for example, are usedto monitor the levels of an analyte in a test subject's bodily fluidover time, the added variable of using multiple subjects is introducedinto the trial. When, however, a single test animal can be used as itsown control over a course of time, a more accurate and beneficialpreclinical trial can be performed.

In some embodiments a method of automatically monitoring patientcompliance with a medical treatment using a fluidic device comprisesallowing a sample of bodily fluid to react with assay reagents in afluidic device to yield a detectable signal indicative of the presenceof an analyte in said sample; detecting said signal with said fluidicdevice;

comparing said signal with a known profile associated with said medicaltreatment to determine if said patient is compliant or noncompliant withsaid medical treatment; and notifying a patient of said compliance ornoncompliance.

Noncompliance with a medical treatment, including a clinical trial, canseriously undermine the efficacy of the treatment or trial. As such, insome embodiments the system of the present invention can be used tomonitor patient compliance and notify the patient or other medicalpersonnel of such noncompliance. For example, a patient taking apharmaceutical agent as part of medical treatment plan can take a bodilyfluid sample which is assayed as described herein, but a metaboliteconcentration, for example, detected by the reader assembly may be at anelevated level compared to a known profile that will indicate multipledoses of the pharmaceutical agent have been taken. The patient ormedical personnel may be notified of such noncompliance via any or thewireless methods discussed herein, including without limitationnotification via a handheld device such a PDA or cellphone. Such a knownprofile may be located or stored on an external device described herein.

In some embodiments noncompliance may include taking an improper dose ofa pharmaceutical agent including without limitation multiple doses andno doses, or may include inappropriately mixing pharmaceutical agents.In preferred embodiments a patient is notified substantially immediatelyafter the signal is compared with a known profile.

A patient or subject of a clinical trial may forget to take a bodilyfluid sample as described herein. In some embodiments a method ofalerting a patient to test a sample of bodily fluid using a fluidicdevice as described herein comprises providing a protocol to be run onsaid fluid device, said protocol located on an external device,associated with said patient, and comprising a time and date to testsaid sample of bodily fluid; and notifying patient to test said bodilyfluid on said date and time if said sample has not been tested. In someembodiments a patient can be notified wirelessly as described herein.

A patient may be provided with a fluidic device or devices whenprocuring a prescription of drugs by any common methods, for example, ata pharmacy. Likewise, a clinical trial subject may be provided with suchdevices when starting a clinical trial. The patient or subject's contactinformation, including without limitation cell phone, email address,text messaging address, or other means of wireless communication, may atthat time be entered into the external device and associated with thepatient or subject as described herein, for example, in a database.Software on the external device may include a script or other programthat can detect when a signal generated from a detection device has notyet been sent to the external device, for example at a given time, andthe external device can then send an alert notifying the patient to takea bodily fluid sample.

In some embodiments the present invention provides a method of assessingthe reliability of an assay for an analyte in a bodily fluid with theuse of a fluidic device. The method comprises the steps of a) providinga system, said system comprising a fluidic device, said fluidic devicecomprising a sample collection unit and an assay assembly, wherein saidsample collection unit allows a sample of bodily fluid to react withreactants contained within said assay assembly, for detecting thepresence of an analyte in a bodily fluid from a subject, and a readerassembly for detecting the presence of said analyte; and b) sensing witha sensor a change in operation parameters under which the systemnormally operates.

In some aspects a sensor may be present either in the fluidic device,the reader assembly, both, or in some cases it may be advantageous toinclude a sensor in the packaging in which the fluidic device and/orreader assembly are packaged. The sensor can, for example withoutlimitation, detect temperate or pressure changes that may provide for aninaccurate analyte concentration calculation. For example, if thetemperature of reagents stored in said fluidic device falls outside anacceptable temperature range, this may indicate that the detection willnot be accurate using the then existing calibration and processingalgorithm, for example. Likewise, for example, the pressure in the pumpin the reader assembly may fall outside an acceptable range. In someembodiments a moisture sensor is provided to detect the presence ofmoisture in the cartridge before the assay begins. In some embodimentsthere may be thiosyanate in one layer of the fluidic device and ironsalt in another layer, wherein a dye is formed when these are mixed,whereby the dye is a visual indication of the presence of moisture.

In some disposable systems, particularly in those where sampleacquisition is performed by the patient or end user, measurement errorsare not uncommon. Significant errors due to, for example, patienthandling of the sample, could be due to the sample collection method. Apatient may not collect the correct volume of the sample, the collectionmay not be performed at the appropriate time, or the sample may not behandled in an appropriate manner, thus compromising the sampleintegrity. It may be advantageous when using a disposable system inwhich the patient controls the initial sample collection and handling toutilize methods for minimizing the consequences of such errors by, forexample, either alerting the patient to repeat the test or usecalibration steps to compensate for such errors.

There is therefore a significant need for methods that would improve thecalibration in hand held or disposable assay units, particularly inthose units where the sample and reagent volumes are in the microliterand nanoliter ranges, where maintaining a controlled temperature isimpractical, where the sample is not “clean” such that errors are causedby interfering substances, such as hematocrit, for example, or where itis difficult to maintain the desired conditions such as temperature orreagent quality, including the appropriate sample volume and handling bythe user.

Immunoassays have a characteristic response similar in form to thewell-known Scatchard binding isotherm (Bound/Maximum Bound (B/B0)=LigandConcentration/(K+Ligand Concentration) where B is the amount of thelabeled analyte bound to a solid phase when analyte is present, B0 isthe amount bound when no analyte is present and K is the dissociationconstant. The mathematical form of such assay responses is hyperbolic.

Results of immunoassays of the types described above are typicallyanalyzed using the known (ln-logit) or (log-logit) functions, in whichthe assay label (for example in a two-step process, alkalinephosphatase-labeled analyte) bound to a solid phase when analyte ispresent in the assay (“B”) is compared with the amount bound when noanalyte is present (“B0)” to provide the ratio B/B0. Then, the “logit”function (logit=Log [(B/B0)/(1−B/B0)]) is plotted against Log (AnalyteConcentration) resulting in a straight line. (Natural logarithms canalso be used instead of logarithms to base 10). The slope and interceptof this plot can be used to derive simple equations that permit thecalculation of (a) assay signal as a function of analyte concentration,or (b) analyte concentration as a function of assay signal. An exampleof such analysis is shown in FIG. 21 using Thromboxane as the analyte ofinterest. The best fit to the data is given by Equation 1:Signal=(A−D)/(1+(Analyte conc./C)̂B)+D [Equation 1], where A is thesignal at zero analyte concentration, D is the signal at infiniteanalyte concentration, C is the analyte concentration reached at asignal level half way between A and D, and B is a shape parameter. Therelationship between analyte concentration and signal is given by:Analyte concentration=C*((((A−D)/(Signal−D)−1)̂(1/B)) [Equation 2], whereA, B, C and D are identical to the parameters used in Equation 1.

It is possible to compute errors that occur from mis-calibration usingthe equations described herein above. (The Analyte Concentrationfunction from Equation 2 is differentiated with respect to eachpotential variable A, B, C, D and Signal). Estimates of the differencebetween the ideal value of the variable and the actual value in thesystem are used as Δ values in the calculation(Δ(concentration)=(d(Concentration)/d(Param.))*ΔParam). Errors incalibration are reflected in erroneous values of A, B, C and D. Each ofthese parameters is influenced by a different factor. For example,temperature effects on calibration of immunoassays will have thestrongest impact on the A, C and D parameters of the lax-logitcalibration, while likely having a minimal impact on the shape parameterB. The detected signal, which in turn can be used to determine theanalyte concentration, is biased by one or more of the following readerassembly and fluidic device characteristics: optics used in theinstrument for signal measurement temperature control; most chemicalprocesses are highly temperature sensitive, including enzyme reactions,and equilibrium between antigens and antibodies; timing of assay steps;calibration relative to an “ideal” instrument; the inability of thepatient to manually recalibrate the fluidic device when used; dimensionsof the fluidic device; volume of the assay assembly and its shape; fluidmovement within the device; timing and uniformity of fluid movement;efficiency in mixing (most assay methods used in disposables and employmicrofluidics would involve some mixing). The following reagentvariations can also contribute to a biased detected signal: reagentquantity; reagent dissolution (if it is in dry form); changes inactivity of reagents following manufacture (instability) (This isparticularly important for “distributed systems” where the disposableuseful life is typically determined by reagents which can, for example,lose 20% of their activity. If they can be used without significantlycompromising assay performance, the shelf-life of many expensivedisposables could be extended several fold and severe constraints ondisposable storage (refrigeration and the like) can be relaxed). Inaddition, when calibration is performed at the factory, small errors inthe estimation of the calibration parameters can result in error in thecalculated analyte concentration.

The magnitudes of these calibration errors and consequently errorsintroduced in estimating analyte concentrations can be quitesignificant. FIG. 21 shows the dose-response data for a two-step assayfor Thromboxane. The top curve (Logit.test) in FIG. 22 shows a typical(ln-logit) assay response. When we adjust the level of the highestsignal (A) and the lowest signal (D), shown as “Shift zero signal” and“Shift 100% signal”, respectively, the curves shift as seen in FIG. 22.The corresponding computed values of error in the concentration thatwould be calculated from Equation 2 were large (>20% across the entirerange of the assay) as shown in FIG. 23. In FIG. 22, the signal isnormalized by subtracting the D value from the signal and dividing thedifference by (A−D):(Signal−D)/(A−D). This yields what is usuallydescribed as B/B₀ (the ratio of bound label at a given analyteconcentration to that at zero analyte level). The ln-logit function wasmodified by adding 10% of (A−D) to D or subtracting 10% of (A−D) from Abefore recalculating the normalized signals (corresponding to two typesof significant calibration error (shifting the value of A or Drespectively). At signal levels intermediate between A and D the changemade was adjusted by 10%*(Original signal−D)/(A−D). FIG. 23 shows thatwhen modifications of only 1%*(A−D) were made, and concentration of theanalyte was computed, errors in concentration were still significant atcertain parts of the analyte concentration range.

In a laboratory setting, errors in measuring biochemical parameters ofblood and other bodily fluids due to calibration errors are dealt withusing many known compensation mechanisms. One of the simplest techniquesis to add a known quantity of a trace amount of a radiolabeled analyteand construct a calibration curve based on those readings. Other methodsinclude adding a known amount of a standard to the analyte solution thatneeds to be analyzed. However, such techniques are impractical in adisposable, handheld system for analysis, without particular adaptationof those techniques for dealing with small sample volumes, lack of largeamounts of other solutions (such as buffers), and ability to exerciseprecise controls over the volumes of the samples and their dilutions.

Conventionally, a calibration exercise is performed in parallel withassaying the sample. This is, however, impractical in a self-contained,disposable assay system intended to be compact and inexpensive. Toaddress any calibration challenges that may occur while assayinganalytes using a fluidic device of the present invention, in someembodiments parameters A, or in preferred embodiments A and D, ofEquation 1 described herein above, are measured within the fluidicdevice rather than using manufacturer's values or an external device.The value(s) is compared with the parameter values estimated when thefluidic device was calibrated by the manufacturer. Signal results arethen adjusted using the following equation:Signal_(adjusted)=Signal*(A_(factory calibration)/A_(measured within the assay))and the original calibration equation (Equation 1) is then used tocalculate the analyte concentration. Alternatively, A and D valuesmeasured at the time of assay are substituted for the A and D valuesobtained during factory calibration. Typically the (A/D) calibrationmeasurement would be made in a buffer sample, preferably for eachanalyte (in a multiple analyte assay device), or one analyte only, ifeach assay responds similarly to the various factors that alter thecalibration parameters.

In some embodiments of this invention, the calibration parameters ofEquation 1 are corrected using differential calibration. The followingexample using Thromboxane B2 as the analyte illustrates this approach.Thromboxane B2 (TxB2) (1.25 mg) was dissolved in a mixture ofdimethylsulfoxide (342 μl) and water (342 μl). To this, 5 μl of asolution of 1-(3-(dimethylamino)propyl)-3-ethyl-carbodiimidehydrochloride in water (0.1 g/ml) and 10 μl of a solution ofn-hydroxy-succinimide in water (0.1 g/ml) were added. After 1 hour atroom temperature the resulting NHS-ester of TxB2 was used in thepreparation of TxB2 labeled with alkaline phosphatase (described below)without further purification. Alkaline phosphatase (bovine intestine,Sigma-Aldrich) was dissolved in phosphate-buffered saline at 1 mg/ml. To1 ml of this solution 120 μl of the NHS-ester of TxB2 was added and themixture allowed to react for 1 hour at room temperature. The enzyme-TxB2conjugate was then purified overnight by dialysis against tris-bufferedsaline containing MgCl₂.

Described is an example of a two-step enzyme immunoassay where TxB2 isthe analyte. Samples and mouse monoclonal anti-TxB2 (15 μl of CaymanChemical Kit Catalog number 10005065, appropriately diluted into AssayDesigns buffer) were added to 384-well plates to which anti-Mouse IgGhad been immobilized ((Becton Dickenson 356177)). The sample was 30 μlof plasma diluted 1:4 with assay buffer (Assay Designs Correlate-CLIA™kit 910-002) and supplemented with known concentrations of TxB2. Othertypes of sample (for example TxB2 dissolved in assay buffer) can besubstituted.

Plates were covered to prevent evaporation and incubated at roomtemperature with gentle mixing (100 rpm) on an orbital shaker for 12hours. The contents of the wells were then removed by aspiration.Thromboxane-labeled with alkaline phosphatase (25 μl diluted 1:1500 withassay buffer) was added and incubated at room temperature for 2 minutes.The contents of the wells were removed by aspiration and wells washedthrice with 100 μl wash buffer (from the Assay Designs Kit 910-002).

Enzyme bound to the wells was then measured by addition of 40 μlLumiphos™ 530 substrate solution which contains(4-methoxy-4-(3-phosphate-phenyl-spiro-[1,2-dioxetane-3,2′-adamantane])).Incubation was allowed to proceed for 1 hour with orbital mixing and theluminescent product measured in a Molecular Devices MD5 Spectrometer(0.5 second integration time).

FIG. 21 shows the typical assay dose-response data for a two-step assayfor TxB2. Using Equation 1, the parameters A, B, C and D are fitted tothe curve shown in FIG. 21. As described herein, even small changes invalues of the parameters A and D can have a significant impact on themeasured concentration. Thus, any errors in computing A and D aremagnified in the estimated analyte (TxB2) concentration. This concept isillustrated in FIGS. 22 and 23, where even a 1% change in (A−D) resultedin significant errors in estimating TxB2 concentrations in the samples.In FIG. 22, the signal is normalized by subtracting the D value anddividing the difference by (A−D) viz: (Signal−D)/(A−D). This calculateswhat is commonly described as B/B0 (the ratio of bound label at a givenanalyte concentration to that at zero analyte level). The (ln-logit)function was modified by adding 10% of (A−D) to D or subtracting 10% of(A−D) from A before recalculating the normalized signals (correspondingto two types of significant calibration error (shifting the value of Aor D respectively). At signal levels intermediate between A and D, thechange made was adjusted by 10%*(Original signal−D)/(A−D). FIG. 23 showsthe computed errors in estimating the analyte concentrations for a 1%error in estimating A and D. As can be seen for the low analyteconcentrations, the errors are pronounced even for small errors in thecalibration parameters A and D.

FIGS. 24-27 illustrate an embodiment of this invention where the samplecontaining an unknown analyte concentration is spiked with a knownconcentration of the analyte to minimize calibration errors. Spiking canbe achieved by a variety of methods, for example, by incorporatinganalyte in known quantities to the assay well during manufacture of thefluidic device. Separate spike wells could also be accommodated in thefluidic device described herein. FIG. 24 shows calibration usingdifferences between signal response between unspiked and spiked samples.The amount of the spiked analyte is indicated by ×2 and the original(endogenous concentration in the sample) is denoted as originalconcentration or ×1 (pg/ml). The difference in signal between unspikedand spiked sample is plotted against the signal for the originalconcentration for various amounts of known amount of analyte (spike)introduced into the sample. The (ln-logit) parameters (for the top curvein FIG. 24) are shown in Table 1.

TABLE 1 Original Calibration Parameters for Data Shown in FIG. 24 A3.37E+04 B 1.01E+00 C 2.10E+02 D 3.56E+03

The data shown in the top curve in FIG. 24 were used in a recalibrationexercise by calibrating against the difference in signal for eachoriginal concentration level and each level spiked with 200 pg/mlanalyte. Equation 3 shown below was empirically derived and is useful incalculating the original endogenous concentration of analyte. Thebest-fit parameter values in Table 2 were computed by minimization ofthe sum of the square of the differences between target and calculatedanalyte values. Concentration=C*((A−D)/((Signal−D)̂(1/B))+E [Equation 3].

TABLE 2 Calculated Parameter Values for 1-point Spike Calibration A1.20E+02 B 1.996189 C 292.7824 D −0.14393 E −287.931

This calibration was verified as shown in FIG. 25 (log scale) and FIG.26 (linear scale). Note the regression equation was calculated for datain linear form. The formula resulted in near perfect results.

The results of one embodiment of this invention are shown in FIG. 27,where the extent of the recovery of the spike signal is used to correctfor the concentration of the value of the unspiked sample. This methodhas the advantage that changes in the parameter C in the (ln-logit)equation due to, for example, reagent instability, are accounted for.The method involves the following steps: calculate ×1 (endogenousconc.), and ×2 (spike conc.) using original calibration; calculaterecovery of spike as % (×2−×1)/spike [Equation 4]; correct ×1 byrecovery factor: (×1*100/Spike recovery) [Equation 5].

This was tested with the calibration curve shown in FIG. 24 and theoriginal calibration parameters of Table 1. As shown in Table 3, it waspossible to use spike concentration values from 100-500 pg/ml and Cvalues that varied from 500 to 50 such that the actual signalscorresponding to the modified C values were changed very significantlyfrom what had been the case with the original C value and the spikerecovery (calculated with the original C value ranged from 42-420%respectively, yet the recovery of the unspiked sample (once correctedfor the recovery of the spike) was 100% over the entire calibrationrange. This effect is graphically illustrated in FIG. 28, where the Cparameter is varied between 50 and 500 (a ten fold range), but thecorrected values for the analyte concentration (×1) accurately reflectsthe expected analtye concentration.

TABLE 3 Effects of changes in the C parameter on spike and originalanalyte recovery at two original concentration levels: x2 x1 x1 x2recovery recovery C Pg/ml S (x1) pg/ml S (x1 + x2) % % 500 100 2.88E+04500 1.73E+06 42 100 210 100 2.40E+04 500 1.13E+04 100 100 50 1001.36E+04 500 5.83E+03 420 100 500 316 2.21E+04 500 1.50E+04 42 100 210316 1.56E+04 500 9.66E+03 100 100 50 316 7.61E+03 500 5.25E+03 420 100500 100 2.88E+04 200 2.25E+04 42 100 210 100 2.40E+04 200 1.60E+04 100100 50 100 1.36E+04 200 7.80E+03 420 100 500 316 2.21E+04 200 1.84E+0442 100 210 316 1.56E+04 200 1.22E+04 100 100 50 316 7.61E+03 2006.16E+03 420 100

In Table 3, ×1 is the endogenous concentration and ×2 is the spikeconcentration; S is the signal level corresponding to the designatedanalyte concentration; ×2 recovery is the apparent recovery of ×2 and ×1recovery is calculated (using Equation 5) after compensating for ×2recovery (using Equation 4).

The spike level must be carefully chosen. The optimal level will be acompromise between the operating range of the assay and the likely rangeof concentrations of samples. If it is too low, the change in signalcaused by the spike will be too small to be reliably measured. If it istoo high, the assay response will be too shallow to reliably measure thespike. The ideal spike level would change the measured signal by muchmore than the standard deviation in the signal. In the above example,the assay range had been adjusted to make measurements for sample withconcentrations in the range of about 0 to about 500 pg/ml and spikes ofabout 200 to about 1000 pg/ml would likely be useful.

In some embodiments the following various guidelines for choosing spikelevels can be followed: spikes should change the observed signal acrossthe desired range by at least 10%; spikes should be in the same range asthe anticipated mid range of sample concentrations; spikes should beless than about three times the original C value. Note that the usefulpart of the dose-response is from about 0.2*C to about 5*C.

The following example illustrates the estimation of endogenous TxB2concentrations using spike recovery. Two citrated human plasma sampleswere analyzed by the two-step assay. Aliquots of the samples were alsosupplemented (spiked) with known concentrations of TxB2 prior to assay.Some samples were also supplemented with indomethacin (0.1 mM) and/orEDTA (5 mM). Samples were stored either flash-frozen then thawed orrefrigerated unfrozen prior to assay. These procedures generated a setof samples with various original endogenous concentrations (storage andfreezing and thawing tends to cause platelet activation and productionof TxB2; indomethacin inhibits TxB2 production).

The results of the above experiment are shown in FIG. 27. Sample 5A wasknown to have a very low TxB2 concentration (estimated to be <10 pg/ml).When the dose-response of the assay in sample 5 was used to calibratethe assay, the concentration was assumed to be zero. Dose responses forthe other samples 4A, 4N, 5N were then plotted and it was observed thattheir response corresponded to higher concentrations of TxB2 and couldbe fitted to the 5N response by moving each to the left (in thedirection of lower concentration) by an amount corresponding to removinga certain fixed TxB2 concentration from each the known spike levels. Allthe samples had responses that were almost identical in shape to that ofsample 5N. When the curves fitted as closely as possibly to the A5curve, the concentration of TxB2 notionally removed corresponds to theestimate of the TxB2 concentration in the sample.

The original data of FIG. 27 were represented in FIG. 29 by the best fit(ln-logit) approximation. The Solver function in Microsoft Excel wasused to compute a value of TxB2 that caused the A5 response toapproximate that of the sample N5. As can be seen, this generated a goodfit and the computed value (471 pg/ml) is an estimate of theconcentration difference between TxB2 levels in the two samples.

In another embodiment of our invention a single point can could be used(all the points fit closely to the calibration curve, so any singlepoint could have been used) rather than a multi point spike that wasillustrated in the earlier FIGS. 24-27. The following experimentillustrates this concept. Two plasma samples were spiked to many levelsof TxB2 and assayed by the two-step method. Assays were calibrated usingbuffer calibrators rather than plasma-based materials. Results arepresented in FIG. 30. Plasma was analyzed as described earlier. Data inFIG. 30 are plotted on a log scale. The concentration of unspikedsamples was calculated from the calibration and the concentration ofspiked samples taken as “endogenous+spike.” Results are plotted only forthe spiked samples. As can be seen, there was desirable correlationbetween the calculated and known values over the range of about 50 toabout 10,000 pg/ml. When recovery was estimated for spikes in the rangeabout 40 to about 2,500 pg/ml, the correlation was 99.7%.

Spike recovery method for correcting the calibration parameters areuseful for compensating temperature effects on immunoassays inself-contained disposable analytical systems, also some times referredto as handheld analytical systems or assay systems. As is well known,instabilities in temperature during an assay introduce significanterrors in the estimated analyte concentration. Temperature effects oncalibration of immunoassays have the strongest impact on the A, C and Dparameters of the (ln-logit) calibration. It is likely that the B(shape) parameter is minimally affected by temperature changes. As shownabove, the spike recovery method can correct for errors introduced inthe C parameter and hence could be an excellent approach for correctingtemperature induced errors in computing the calibration parameters ofthe (ln-logit) equation. Similarly, normalizing signal levels to thezero analyte calibrator level, as described earlier, can compensate forerrors in the A and D parameters, which are again negatively influencedby temperature changes.

Internal calibration and/or spike recovery means of calibration havesignificant advantages over conventional factory-calibration methods.One obvious advantage is that two quantities of assay-relatedinformation are used to compute the assay result rather than one, whichimproves the reliability of the assay. A second advantage is that thisapproach compensates, to a large extent, reagent instability. Anotheradvantage is that several instrument, assay environment, and proceduralvariables are factored into the assay results.

Other uncontrolled changes in system response, besides temperate change,can also negatively impact the computed A and D parameters. For example,FIG. 31 shows the time course of the signal generation during an assay.To correct for these errors, one embodiment of the claimed invention isto compare assay signals B in a fluidic device with the B0 signal so toeliminate errors due to variation of the absolute value of assay signalsdue to uncontrolled changes in system response. This concept wasverified by the following experiment.

A competitive immunoassay for TxB2 was set up using the protocoldescribed in Assay Designs Product Literature for their correspondingCorrelate-CLEIA kit (catalog 910-002). An alkaline phosphatase conjugatewas prepared as described earlier and was diluted 1:112,000 andsubstituted for the kit conjugate. A and D parameters are thecalibration parameters used in the (log-logit) fit to the assayresponse. Best fit values were obtained at each time point. Note that atzero time the A and D parameters are not measured, but all signal valueswould be (are known to be) zero. The ratio D/A was multiplied by 1e6 soas to be presentable on the same scale. The A and D values when plottedagainst time vary significantly, particularly the A value (zeroanalyte). As seen from the straight line with practically zero slope,the scaled D/A remains constant over the time span.

The above experimental data were then analyzed by normalizing the assaysignal (B) to signal at zero analyte concentration (B0). Using thisnormalized signal (B/B0), (log-logit) best fits were obtained for eachtime point and averaged. Concentrations of analyte were computed usingthese calibration parameters for each time. FIG. 32 shows the derivedconcentrations that were plotted against the A parameter derived foreach individual time point. Each line corresponds to different analytelevels (pg/ml) ranging from about 39 to about 10,000 pg/ml. As can beseen from FIG. 32, although signal values changed by about 2-fold duringthe course of the experiment, the derived analyte concentration wasessentially constant over the analyte concentration spanning a range ofabout 39 to about 10,000 pg/ml. The variation of calculatedconcentration was computed and found to average only 2.7% over thecalibration range of 39-625 pg/ml (which spans most of the range).

A calibration spike can be enabled by adding analyte to the antibody (orother solid phase capture agent) during manufacturing, and then drying.subsequently adding analyte to the appropriate well during manufacturing(then drying), or adding analyte to a portion of assay buffer which isthen routed to the appropriate well. Methods 1 and 2 have a risk whichis that the spiked analyte could be flushed from the well as sample orbuffer enters. This may be handled in one of several ways such asrelying on the tightness of the antigen: antibody interaction for thebrief time the well is subject to flowing sample or buffer (which exitfrom the well), or careful management of liquid flow and placing thespike well as that most distal to the incoming liquid (last well to fillhas the least flow through).

Errors in measuring analyte concentrations could also be due tovariability in the pre-analysis phase. The primary cause of this type oferrors is due to the patient collecting an incorrect volume of sample orwhere the sample integrity has been compromised. Errors due to incorrectsampling volume can by corrected by a variety of means. One method is tomeasure the volume of the sample during a pre-processing step. If themeasured volume is significantly different from the expected volume, thepatient could be instructed to provide a new sample. This could beaccomplished by, for example, the wireless communication with theexternal device as described herein. Alternatively, the analyticalmethods or algorithms on the external device could be recalibrated tocompensate for the change in the sample volume. The recalibration couldbe using any of the standard calibration techniques or the modificationsto the calibration process, which have been described herein.

The following is a description of one embodiment of a method fordetermining the accuracy of the volume of the sample provided to thesample collection unit of a fluidic device described herein. The samplecollection unit can be lined with conductive elements spaced apart atknown separations similar to the graduations on a measuring cylinder orjar. The location of each conductor can correspond to a specific samplevolume. As fluid comes into contact with the conductor, the measuredconductivity of that conductor would be markedly increased. Byidentifying the highest placed conductor that has undergone theconductivity change, the volume of the sample in the sample collectionunit can be computed.

Alternatively, if the sample volume has to meet a minimum, a conductiveelement could be placed at the appropriate level in the well. When thecassette is introduced into the handheld (or the sample holder isintroduced in the analytical system), thereby the patient has indicatedthat she has completed the sampling process, and if the conductivity ofthe sensor remains at the baseline level, it could be easily concludedthat the patient has not provided the required sample volume. Thepatient could be given the appropriate feedback such as replacing thesample or replenishing it. Alternatively, the back-end server orcomputer at the network headquarters could be informed of the issue andappropriate corrective measures taken. An alternative to the electricalsensing for the correct volume could be using known optical sensingmeans.

Sample integrity could be affected by many factors, some intrinsic tothe patient and some that are extrinsic. Following are some of thesources of errors in sample integrity: (i) mixing of interstitial fluidwith blood; (ii) variability in the hematocrit concentration; (iii)hemolysis; and (iv) activation of platelets and sample clotting.

Occasionally, interstitial fluid may leak from a finger-puncture woundand could mix with blood. Alternatively, if the patient had liquid onher hands due to washing prior to obtaining a blood sample, such liquidcould also mix with blood plasma. Both fluids mentioned, above,interstitial fluid and wash liquid, contain no red cells and would mixwith the blood plasma. When the amount of interstitial fluid is large sothat the effective hematocrit is very low, the measured concentration ofthe external standard (fluorescein) would be low. This signal could beused to conclude that the sample is inappropriate for analysis and thatit could lead to incorrect results. When blood is contaminated by water(which has low conductivity), it would be possible to detect this bymeasuring the conductivity of the fluid part of the sample (blood plasmahas a characteristic high conductivity not subject to variation fromday-to-day or individual-to-individual). If the measured conductivity ofthe sample is lower than the plasma conductivity, it is likely that thesample has been contaminated.

Errors could also be due to incorrect operation of the instrument andmeans of detecting and compensating those errors are described below.One source of error could be that the disposable is not properlyaccommodated in the handheld system. Having a sensor detect and reportthe proper mating of the disposable in the handheld would be one meansof avoiding this problem. Another source of errors is from the fluidicsystem, where there may be an issue with where the sample is applied inthe sample well and the volume of the applied sample. This could againbe addressed by the use of appropriate sensors which detect theapplication of a sample and report on the adequacy of the volume that isapplied. Other fluidics related problems could be blocked channels,insufficient reagents, bubbles, etc., all of which again could bedetected and reported by the use of appropriate sensors.

In some embodiments any of the errors described herein can be measuredusing sensors located on either the fluidic device or the readerassembly. In some embodiments an error messages could be displayed on anLCD screen in the reader assembly using the processing power of themicrochip on the handheld. Alternatively, a signal from the sensorscould be communicated to the external device which can then relay anerror message to either the reader assembly or a third device such as aPDA or cell phone. Such action could be a message communicated to thepatient in the form of an audio, video or simple text message that thepatient could receive. In some embodiments the external server cantransmit corrected calibration parameters to the reader assembly tocompensate for any of the errors described herein.

In yet another embodiment, after the identifier is detected by anidentifier detector as described herein to determine, for example, aprotocol, if a signal transmitted by a sensor doesn't match the expectedvalue for the sensor signal, then the external device can transmit apre-programmed alert based on each cartridge bar code and sensed signalto either, for example, an LCD display on the reader assembly or to ahandheld device, to take a designated action. Nonlimiting examples oferror alerts, the problems they indicate, and required action to betaken are, for example:

Error Code Symbol Problem Action Er1 Thermometer Temperature out of Waituntil Temp >10 or <35 C. range Er2 Blood drop Blood sample too small Ifdetected w/in 15 minutes of first sample add more blood, other wise usenew cartridge Er3 Battery Power disruption Do not start test until powerresumes Er4 Bar code symbol Cartridge expired Run test on a non expiredcartridge Er5 Line through fluidic device Cartridge already used Runtest on a new cartridge Er6 Phone receiver No Cell Phone Do not starttest until in coverage coverage area Er7 Line through a box Readermalfunction Call Theranos Er8 Bottle with a “C” in the label Calibrationoverdue Run Calibration standard, then run test

After the identifier detector detects the identifier to determine aprotocol and any sensed signals are detected and either patientnotification is complete or calibration parameter are updated, thefluidic device calibration can occur, followed by the appropriate assay.

Despite the corrective actions described here, the generated analyteconcentrations values could still be erroneous. For example, the actualanalyte concentration could be well outside the expected range, and thusthe calibration parameters used may be incorrect. Values which areunlikely, impossible or inconsistent with prior data for a particularlypatient could be flagged and subjected to a software review. Values withsuspect accuracy can be communicated to the appropriate decision maker,such as the patient's physician.

The concept of the reference therapeutic index (TI) and how it iscomputed is illustrated in FIGS. 33 and 34. A TI is computed from aretrospective analysis of many measured parameters, including the bloodconcentrations of drugs of interest, their metabolites, other analytesand biomarkers in blood that change concentrations due to the drugs thepatient is consuming, physiologic parameters (such as blood pressure,respiratory rate, body temperature, heart rate, etc.), and clinicalparameters that indicate disease progression (such as angina, stroke,infarct, etc.). Typically, many serial measurements would be made forthe many treated patient and corresponding controls (unmedicated orplacebo treated). The clinical parameter would be an “outcome parameter”(OP). The other measured parameters can be “input parameters” (IP).

For the retrospective analysis and TI computation, data from manysubjects and their respective output and input parameters, includingsubject's relevant details such as height, weight, race, sex, familyhistory, etc., would be populated in a database. Each candidate outcomeparameter (stroke, infarct, angina, death, etc.) will be subject tomultiple regression analysis against input parameters.

The multiple regression analysis is performed for each candidate OPversus all available IPs. Database columns are constructed by using eachIP, each IP̂2, and all cross-terms (IPi*IPj). The analysis is thenperformed using the equation:

OPi=(a*IP1+b*IP2+ . . . n*IPn)+(aa*IP1̂2+bb*IP2̂2+ . . .+nn*IPn̂2)+(aaa*IP1*IP2+bbb*IP1*IP3+ . . . +nnn*IPn−1*IPn), where a . . .n, aa . . . nn, aaa . . . nnn are arbitrary constants.

Multiple regression analysis establishes the best fit to the equationand indicates which IPs are strong candidates for inclusion. Weaklycorrelated IPs are dropped and the analysis repeated until eachcandidate OP has an optimal relation to the remaining IPs. Thetherapeutic index will then have the form:

TI=a*IP+cc*IP3̂2+nnn*IP3*IP5+ . . .  (Equation 6).

FIG. 34 illustrates the computation of a TI and the use of the TIconcept for determining therapeutic efficacy (the therapeutic index isalso indicated by the term efficacy index). The example illustrated inFIG. 34 shows the time course of successful drug therapy of a diseasestate (such as atherosclerosis) that is indicated by three biochemicalanalytes represented by parameters A, B and C. The disease is treated(with for example a Statin) starting on day zero.

Parameters A, B and C are measured daily using an ambulatory system asdescribed herein. At the outset, relative to “ideal levels”, Parameter A(for example LDL-cholesterol) is elevated, Parameter B (for exampleHDL-cholesterol) is low and Parameter C (for example, alanineaminotransferase, an indicator of liver damage) is normal. Allparameters (A, B, C) are presented normalized to their respective ideallevel. As therapy proceeds, the drug causes the levels of A and B toapproach normal values but at different rates. Analyte C remains normalindicating the drug is not causing liver damage. The relative risk of anoutcome for the patient is represented by an initially unknown TI. Asdescribed above, TI is a surrogate to the outcome parameter thatreflects the physiological functions of the patient (blood pressure,etc.) or other pre-identified factors in a patient record and can beindicative of improvement in the patient's condition. We further assumethat parameter TI is influenced by parameters A and B. In certain cases,at the beginning of the study this relationship remains to bedetermined.

Data from the monitoring system (device input) and the patient input areanalyzed by multiple regression of TI and measured values A, B and C, asdescribed above. In the example shown, these data are analyzed usingmultiple regression analysis, which fits parameter TI as a function ofparameters A, B, C and their squares and the pair-wise cross terms (A*B,etc.) As shown in FIG. 35, for the simulated values shown in FIG. 34, anexcellent fit was obtained (R̂2=0.99) when all parameters were included.It is evident from inspection of the fit that most of the parameters canbe eliminated leaving only A and A*B. When this is done the fit is stillvery good (R̂2=0.95).

The multiple regression derived function is not identical to the basefunction which generated the first candidate TI data, but works well tocompute an estimate of TI from (typically fewer) measured parameters,prior to clinical validation, if necessary. The appropriate thresholdlevels of TI, or the optimum TI is termed as TI_(ref) (or “actionthreshold value”.) Expert review can then determine the optimumtherapeutic index for that particular patient or patient class. If thecomputed TI exceeds the preset TI_(ref), appropriate action can betaken. An appropriate action could be alerting the physician, stoppingthe medication or the like. As can be understood, the appropriateTI_(ref) for a patient would be decided based on the healthcareprovider's judgment for that individual patient. The form of the TI isderived as a one time exercise using expert analysis of the data setderived from clinical studies and/or existing clinical information.

Once the TI_(ref) is identified, then the use of this parameter isillustrated in FIG. 36. Methods of measuring drug, analyte and biomarkerconcentrations and conducting a two-way communication with a databaseusing a fluidic device and reader assembly are described in detailherein. The time course of various measured and computed parameters areshown in FIG. 36. The curve indicated CBX Dose illustrates the timecourse of a drug that is taken on a regular basis. The plotted valuesare normalized to what would be considered as “ideal levels” for thatmeasurement. For example, if the expected ideal blood concentration ofCBX is 100 ng/ml and if the measured concentration in blood is 100ng/ml, the parameter value is 1.0 (with no offset) for CBX. Similarly,the concentrations of CXB, a metabolite of CBX, biomarkers Tx-M andPGI-M, which vary in response to the concentrations of the drug and thedisease state, are also normalized to their ideal values and plotted.All the drug, analyte and biomarker concentrations could be measuredusing a system as described herein. As explained above, the TI_(ref) forthis particular patient is plotted on FIG. 36 as a flat line. Using theparameter values (a . . . n, aa . . . nn, aaa . . . nnn) of Equation 6and the measured input parameters (IP), the current TI for the patientis calculated. If the computed TI exceeds the TI_(ref) value, then analert can be generated. The alert could be targeted to the patient'shealthcare provider, who in turn can take the appropriate action. Anappropriate action could be to watch the patient closely for otherclinical indications and/or alter the dosage and drugs the patient istaking.

FIGS. 36 and 37 illustrate the concept as to how when the computed TIexceeds the TI_(ref) a proactive action could avert an ADR. In FIG. 36,the patient's TI exceeded TI_(ref) about day 15. The patient ismonitored closely and as the TI values continue to increase after day30, the physician intervenes and reduces the dosage. This action startslowering the TI for the patient and ultimately retreats to an acceptablelevel about day 60.

One or more individuals or entities that are involved in the care of thepatient (nurses, physicians, pharmacist, etc.) can be alerted when thecomputed TI exceeds the TI_(ref) so that they could take the appropriateaction. Additionally, trends can be discerned and appropriate actiontaken before a TI reaches a particular value.

In some embodiments many different analytes can be measured andconstrued as input parameters, IPs, while computing the TI. Suchanalytes that may be used are described herein. Additionally, the can beexpanded or modified depending on the disease area as well. Theappropriate list of parameters relating to certain diseases and drugtreatments, for example, cancer and infectious diseases and patient onNSAIDS, are disclosed herein.

In another aspect of this invention, the TI is calculated usinginformation derived from the patient's biological sample and patientinformation that is non-drug related, the device input. For example, inan ambulatory setting, information relating to concentration of drug,metabolite and other biological markers can be detected in blood asdescribed herein. The patient can also input many non-drug relatedpersonal parameters. This “patient input” can relate to the patient'spersonal information, for example, height, weight, gender, dailyexercise status, food intake, etc. The patient input could also beprovided by the patient's healthcare provider. An example of a patientinput parameter and the input means is shown in FIG. 38.

In some embodiments the device input and patient input are used tocompute the TI. A reference TI for the patient is already known usingretrospective analysis of the data contained in the database. Informulating the TI using multiple regression analysis, the parameterssuch as those shown in Equation 6 are used. The same parameters are thenused with the device input and patient input to compute the TI.Comparing the TI to the TI_(ref), it is possible to determine theefficacy of the therapy. If the TI falls within a pre-determined rangeof TI_(ref), then the treatment is considered to be efficacious. Valuesbelow that range indicate that the treatment is ineffective and valueshigher then the range are considered to be undesirable and could lead toadverse events.

Another example illustrates the implementation of this invention forstudying the efficacy of therapy in diseases where it is difficult tomake frequent measurements and the efficacy of the treatment isdifficult to quantify. An example is determining the efficacy of drugtherapy in children with autism. Frequent sampling and concomitantlaboratory analysis is impractical for children. Abnormalities in bloodconcentrations of certain metals are implicated in autism. Hence,following the blood concentration of certain metals, e.g., zinc, inautistic children might shed light on the efficacy of an intervention.However, it has been reported that lowered concentrations of, say, Zndue to a treatment does not imply that the therapy is working. It is anindicator, but not a definitive surrogate for determining therapeuticefficacy. Computing a TI and comparing it to a reference level wouldbetter indicate the efficacy. This is illustrated in FIG. 39 bysimulating the concentration of various pertinent markers and theirchange due to a drug intervention in an autistic child.

The program can involve monitoring subjects and matched controlindividuals over time for toxic metals, surrogate markers for metals(metallothionein, etc.), and other biochemical markers. Subjects arethose prone to, or afflicted with autism; controls are situation-matchedpeople. It is not mandatory that there be a situation-matched control.The scenario assumes that during the study a significant “event” occurs.Events could be movement into a more or less risky environment orinitiation of therapy. Subjects could be frequently monitored forseveral parameters (device input) using the ambulatory system describedherein. Additional laboratory assays that are not determinable in theambulatory system could be performed at a lower frequency usinglaboratory assays. Additional data such as patient information, localenvironment, use of drugs, diet, etc. would be logged (patient input).Of particular interest to this scenario is information such as exposureto lead, mercury etc.

The time course shown in FIG. 39 envisages an event (initiation oftherapy) at 33 days. The subject who is exhibiting abnormal levels of CPand MT, gradually reverts to normal levels of markers. The TI capturesthe risk or safety level of the subject based on all information. Thestudy will define the best inputs to determine TI.

As described above, TI can be used for determining the efficacy of drugtreatment. A similar approach is also well suited for determining theefficacy of drugs during clinical trials. Additionally, this approachcould be beneficially used to identify sub-groups of patients whorespond well or poorly to a given treatment regimen. The ability tosegregate responders from non-responders is an extremely valuable tool.The concept of using TI can be used not only during a therapeuticregimen, but for performing diagnostic tests to determine, for example,whether or not a patient is in need of a biopsy after a completeexamination of prostate specific markers.

TABLE 4 Exemplary Analyates Liver LDH, (LD5), (ALT), Arginase 1 (livertype), Alpha-fetoprotein (AFP), Alkaline phosphatase, Alanineaminotransferase, Lactate dehydrogenase, and Bilirubin Kidney TNFaReceptor, Cystatin C, Lipocalin-type urinary prostaglandin D, synthatase(LPGDS), Hepatocyte growth factor receptor, Polycystin 2, Polycystin 1,Fibrocystin, Uromodulin, Alanine, aminopeptidase, N-acetyl-B-D-glucosaminidase, Albumin, and Retinol-binding protein (RBP)Heart Troponin I (TnI), Troponin T (TnT), CK, CKMB, Myoglobin, Fattyacid binding protein (FABP), CRP, D-dimer, S-100 protein, BNP, NT-proBNP, PAPP-A, Myeloperoxidase (MPO), Glycogen phosphorylase isoenzymeBB (GPBB), Thrombin Activatable Fibrinolysis Inhibitor (TAFI),Fibrinogen, Ischemia modified albumin (IMA), Cardiotrophin-1, and MLC-I(Myosin Light Chain-I) Pancrease Amylase, Pancreatitis-Assocoatedprotein (PAP-1), and Regeneratein proteins (REG) Muscle tissue MyostatinBlood Erythopoeitin (EPO) Bone Cross-linked N-telopeptides of bone typeI collagen (NTx) Carboxyterminal cross-linking telopeptide of bonecollagen, Lysyl- pyridinoline (deoxypyridinoline), Pyridinoline,Tartrate-resistant acid phosphatase, Procollagen type I C propeptide,Procollagen type I N propeptide, Osteocalcin (bone gla-protein),Alkaline phosphatase, Cathepsin K, COMP (Cartillage Oligimeric MatrixProtein), Osteocrin Osteoprotegerin (OPG), RANKL, sRANK, TRAP 5 (TRACP5), Osteoblast Specific Factor 1 (OSF-1, Pleiotrophin), Soluble celladhesion molecules (SCAMs), sTfR, sCD4, sCD8, sCD44, and OsteoblastSpecific Factor 2 (OSF-2, Periostin) Cancer PSA (total prostate specificantigen), Creatinine, Prostatic acid phosphatase, PSA complexes,Prostrate-specific gene-1, CA 12-5, Carcinoembryonic Antigen (CEA),Alpha feto protein (AFP), hCG (Human chorionic gonadotropin), Inhibin,CAA Ovarian C1824, CA 27.29, CA 15-3, CAA Breast C1924, Her-2,Pancreatic, CA 19-9, Carcinoembryonic Antigen, CAA pancreatic,Neuron-specific enolase, Angiostatin DcR3 (Soluble decoy receptor 3),Endostatin, Ep-CAM (MK-1), Free Immunoglobulin Light Chain Kappa, FreeImmunoglobulin Light Chain Lambda, Herstatin, Chromogranin A,Adrenomedullin, Integrin, Epidermal growth factor receptor, Epidermalgrowth factor receptor- Tyrosine kinase, Pro-adrenomedullin N-terminal20 peptide, Vascular endothelial growth factor, Vascular endothelialgrowth factor receptor, Stem cell factor receptor, c-kit, KDR or Flt-1,KDR, AML, and Midkine Infectious disease Viremia, Bacteremia, Sepsis,PMN Elastase, PMN elastase/α1-PI complex, Surfactant Protein D (SP-D),HBVc antigen, HBVs antigen, Anti-HBVc, Anti-HIV, T-supressor cellantigen, T-cell antigen ratio, T- helper cell antigen, Anti-HCV,Pyrogens, p24 antigen, Muramyl- dipeptide Diabetes C-Peptide, HemoglobinA1c, Glycated albumin, Advanced glycosylation end products (AGEs),1,5-anhydroglucitol, Gastric Inhibitory Polypeptide, Glucose,Hemoglobin, ANGPTL3 and 4 Inflamation Rheumatoid factor (RF),Antinuclear Antibody (ANA), C-reactive protein (CRP), Clara Cell Protein(Uteroglobin) Allergy Total IgE and Specific IgE Autism Ceruloplasmin,Metalothioneine, Zinc, Copper, B6, B12, Glutathione, Alkalinephosphatase, and activation of apo-alkaline phosphatase Coagulationb-Thromboglobulin, Platelet factor 4, Von Willebrand factor disordersCOX inhibitors TxB2 (Cox-1), 6-keto-PGF-1-alpha (Cox 2),11-Dehydro-TxB-1a (Cox-1) Geriatric Neuron-specific enolase, GFAP, andS100B Nutritional status Prealbumin, Albumin, Retinol-binding protein(RBP), Transferrin, Acylation-Stimulating Protein (ASP), Adiponectin,Agouti-Related Protein (AgRP), Angiopoietin-like Protein 4 (ANGPTL4,FIAF), C- peptide, AFABP (Adipocyte Fatty Acid Binding Protein, FABP4)Acylation-Stimulating Protein (ASP), EFABP (Epidermal Fatty Acid BindingProtein, FABP5), Glicentin, Glucagon, Glucagon-Like Peptide-1,Glucagon-Like Peptide-2, Ghrelin, Insulin, Leptin, Leptin Receptor, PYY,RELMs, Resistin, amd sTfR (soluble Transferrin Receptor) Lipidmetabolism Apo-lipoproteins (several), Apo-A1, Apo-B, Apo-C-CII, Apo-D,Apo-E Coagulation status Factor I: Fibrinogen, Factor II: Prothrombin,Factor III: Tissue factor, Factor IV: Calcium, Factor V: Proaccelerin,Factor VI, Factor VII: Proconvertin, Factor VIII:, Anti-hemolyticfactor, Factor IX: Christmas factor, Factor X: Stuart-Prower factor,Factor XI: Plasma thromboplastin antecedent, Factor XII: Hageman factor,Factor XIII: Fibrin-stabilizing factor, Prekallikrein,High-molecular-weight kininogen, Protein C, Protein S, D-dimer, Tissueplasminogen activator, Plasminogen, a2- Antiplasmin, Plasminogenactivator inhibitor 1 (PAI1). Monoclonal those for EGFR, ErbB2, andIGF1R antibodies Tyrosine kinase Ab1, Kit, PDGFR, Src, ErbB2, ErbB 4,EGFR, EphB, VEGFR1-4, inhibitors PDGFRb, FLt3, FGFR, PKC, Met, Tie2,RAF, and TrkA; VEGF Serine/Threoline AKT, Aurora A/B/B, CDK, CDK (pan),CDK1-2, VEGFR2, PDGFRb, Kinase Inhibitors CDK4/6, MEK1-2, mTOR, andPKC-beta GPCR targets Histamine Receptors, Serotonin Receptors,Angiotensin Receptors, Adrenoreceptors, Muscarinic AcetylcholineReceptors, GnRH Receptors, Dopamine Receptors, Prostaglandin Receptors,and ADP Receptors Other Theophylline, CRP, CKMB, PSA, Myoglobin, CA125,Progesterone, TxB2, 6-keto-PGF-1-alpha, and Theophylline, Estradiol,Lutenizing hormone, High sensitivity CRP, Triglycerides, Tryptase, Lowdensity lipoprotein Cholesterol, High density lipoprotein Cholesterol,Cholesterol, IGFR, Leptin, Leptin receptor, and Pro-calcitonin, BrainS100 protein, Substance P, 8-Iso-PGF-2a; GIP; GLP-1

1.-29. (canceled)
 30. A method for programming a device capable ofanalyzing a bodily fluid, comprising: selecting by a prescribingphysician or a drug company at least one threshold value of at least oneanalyte to be sensed by the device, the at least one threshold value ofthe at least one analyte being associated with a particular drug beingor to be taken by the patient or course of treatment for the patient;providing an external storage unit separate from the device, wherein theexternal storage unit contains stored information including the at leastone threshold value of at least one analyte to be sensed by the deviceselected by the prescribing physician or the drug company; reading thestored information stored on the external storage unit into a readerassembly associated with the device; setting the device with the atleast one threshold value for the at least one analyte to be sensed bythe device with the information read by the reader assembly from theexternal storage unit, wherein the at least one threshold value is avalue such that a sensed analyte level beyond the value will cause adisplay to display an alert; sensing the analyte level; and displayingan alert if the sensed analyte level is beyond the threshold value. 31.The method of claim 30, wherein the external storage unit is provided tobe operatively associated with a drug-containing unit.
 32. The method ofclaim 30 or 31, wherein the reader assembly is a bar code reader and theexternal storage unit is associated with a bar code.
 33. The method ofclaim 30 or 31, wherein the reader assembly is an electromagnetic signalreader and the external storage unit is associated with anelectromagnetic signal.
 34. The method of claim 33, wherein the drug isprovided in a closed drug-containing unit and the method furthercomprises opening the drug-containing unit and detecting saidelectromagnetic signal upon opening the drug-containing unit.
 35. Themethod of claim 30 or 31, wherein the reader assembly is a magneticsignal reader and the external storage unit is associated with amagnetic signal.
 36. The method of claim 30, wherein the at least onethreshold value of the at least one analyte to be sensed by the deviceis selected by a prescribing physician for the patient or for a class ofpatients.
 37. The method of claim 30, wherein the at least one thresholdvalue of the at least one analyte to be sensed by the device is selectedby a drug company for the patient or for a class of patients.
 38. Asystem for monitoring a patient, comprising a device and an externalstorage unit provided separately from the device and containing storedinformation set by a prescribing physician or a drug company andincluding at least one threshold value of at least one analyte to besensed by the device, the at least one threshold value of the at leastone analyte being associated with a particular drug being or to be takenby the patient or course of treatment for the patient, wherein thedevice comprises: a sensor for sensing at least one analyte in apatient; a display for displaying processed information concerning thesensed analyte; a reader assembly for reading information from theexternal storage unit; and a processor configured for setting the devicewith the at least one threshold value for the at least one analyte to besensed in accordance with the information read by the reader assemblyfrom the external storage unit, and configured for processing theinformation concerning the analyte and configured for sending theprocessed information to the display, wherein the at least one thresholdvalue is a value such that a sensed analyte level beyond the value willcause the display to display an alert.
 39. The system of claim 38,wherein the reader assembly is a bar code reader.
 40. The system ofclaim 38, wherein the reader assembly is an electromagnetic signalreader.
 41. The system of claim 38, wherein the reader assembly is amagnetic signal reader.
 42. The system of claim 38, wherein the displayis provided proximate the sensor.
 43. The system of claim 38, whereinthe external storage unit is provided to be operatively associated witha drug-containing unit.
 44. The system of claim 38, wherein the readerassembly is a bar code reader and the external storage unit isassociated with a bar code.
 45. The system of claim 38, wherein thereader assembly is an electromagnetic signal reader and the externalstorage unit is associated with an electromagnetic signal.
 46. Thesystem of claim 38, wherein the reader assembly is a magnetic signalreader and the external storage unit is associated with a magneticsignal.
 47. The system of claim 38, wherein the at least one thresholdvalue of the at least one analyte to be sensed by the device is selectedby a prescribing physician for the patient or for a class of patients.48. The system of claim 38, wherein the at least one threshold value ofthe at least one analyte to be sensed by the device is selected by adrug company for the patient or for a class of patients.
 49. A methodfor programming a device capable of analyzing a bodily fluid,comprising: selecting by a patient's healthcare provider or a companysponsoring a drug study at least one action threshold value of at leastone analyte to be sensed by the device, the at least one actionthreshold value of the at least one analyte being associated with aparticular drug being or to be taken by the patient or course oftreatment for the patient; providing an external storage unit separatefrom the device, wherein the external storage unit contains storedinformation including the at least one action threshold value of atleast one analyte to be sensed by the device selected by the patient'shealthcare provider or the company sponsoring the drug study; readingthe stored information stored on the external storage unit into a readerassembly associated with the device; setting the device with the atleast one action threshold value for the at least one analyte to besensed by the device with the information read by the reader assemblyfrom the external storage unit, wherein the at least one actionthreshold value is a value such that a sensed analyte level beyond thevalue will cause a display to display an alert; sensing the analytelevel; and displaying an alert if the sensed analyte level is beyond theaction threshold value.
 50. The method of claim 30 or 49, wherein theexternal storage unit contains at least one threshold value associatedwith a drug-containing unit.
 51. The method of claim 49, wherein the atleast one action threshold value of the at least one analyte to besensed by the device is selected by a healthcare provider for thepatient or for a patient class.
 52. The method of claim 49, wherein theat least one action threshold value of the at least one analyte to besensed by the device is selected by a company sponsoring a drug studyfor the patient or for a patient class.
 53. A system for monitoring apatient, comprising a device and an external storage unit providedseparately from the device and containing stored information set by apatient's healthcare provider or a company sponsoring a drug study andincluding at least one action threshold value of at least one analyte tobe sensed by the device, the at least one action threshold value of theat least one analyte being associated with a particular drug being or tobe taken by the patient or course of treatment for the patient, whereinthe device comprises: a sensor for sensing at least one analyte in apatient; a display for displaying processed information concerning thesensed analyte; a reader assembly for obtaining information from theexternal storage unit; and a processor configured for setting the devicewith the at least one action threshold value for the at least oneanalyte to be sensed in accordance with the information read by thereader assembly from the external storage unit, and configured forprocessing the information concerning the analyte and configured forsending the processed information to the display, wherein the at leastone action threshold value is a value such that a sensed analyte levelbeyond the value will cause the display to display an alert.
 54. Thesystem of claim 53, wherein the reader assembly is a bar code reader.55. The system of claim 53, wherein the display is provided proximatethe sensor.
 56. The system of claim 53, wherein the at least one actionthreshold value of the at least one analyte to be sensed by the deviceis selected by a healthcare provider for the patient or for a patientclass.
 57. The system of claim 53, wherein the at least one actionthreshold value of the at least one analyte to be sensed by the deviceis selected by a company sponsoring a drug study for the patient or fora patient class.